U.S. patent number 8,765,407 [Application Number 12/179,988] was granted by the patent office on 2014-07-01 for l-amino acid producing bacterium and method of producing l-amino acid.
This patent grant is currently assigned to Ajinomoto Co., Inc.. The grantee listed for this patent is Mayu Iyo, Shinichi Sugimoto, Ryo Takeshita. Invention is credited to Mayu Iyo, Shinichi Sugimoto, Ryo Takeshita.
United States Patent |
8,765,407 |
Iyo , et al. |
July 1, 2014 |
**Please see images for:
( Certificate of Correction ) ** |
L-amino acid producing bacterium and method of producing L-amino
acid
Abstract
An L-amino acid is produced by culturing an L-amino
acid-producing bacterium which belongs to the Enterobacteriaceae
family and which has been modified so that the activity of an iron
transporter is increased by enhancing expression of one or more
genes of the following genes: tonB gene, fepA gene, and fecA.
Inventors: |
Iyo; Mayu (Kawasaki,
JP), Takeshita; Ryo (Kawasaki, JP),
Sugimoto; Shinichi (Kawasaki, JP) |
Applicant: |
Name |
City |
State |
Country |
Type |
Iyo; Mayu
Takeshita; Ryo
Sugimoto; Shinichi |
Kawasaki
Kawasaki
Kawasaki |
N/A
N/A
N/A |
JP
JP
JP |
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Assignee: |
Ajinomoto Co., Inc. (Tokyo,
JP)
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Family
ID: |
37888229 |
Appl.
No.: |
12/179,988 |
Filed: |
July 25, 2008 |
Prior Publication Data
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Document
Identifier |
Publication Date |
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US 20090215130 A1 |
Aug 27, 2009 |
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Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
Issue Date |
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PCT/JP2007/051878 |
Jan 30, 2007 |
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Foreign Application Priority Data
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Jan 30, 2006 [JP] |
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2006-020563 |
Sep 7, 2006 [JP] |
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2006-243282 |
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Current U.S.
Class: |
435/69.1;
435/71.1; 435/71.2 |
Current CPC
Class: |
C12P
13/08 (20130101) |
Current International
Class: |
C12P
21/04 (20060101); C12P 21/06 (20060101) |
References Cited
[Referenced By]
U.S. Patent Documents
Foreign Patent Documents
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1 577 396 |
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Sep 2005 |
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EP |
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WO01/53459 |
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Jul 2001 |
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WO |
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WO02/29034 |
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Apr 2002 |
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WO |
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WO2005/064001 |
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Jul 2005 |
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WO |
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WO2006/038695 |
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Apr 2006 |
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WO |
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WO2007/069782 |
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Jun 2007 |
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WO |
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Other References
Postle et al., 1983, PNAS, USA, 80: 5235-5239. cited by examiner
.
Buchanan, S. K., et al., "Crystal structure of the outer membrane
active transporter FepA from Escherichia coli," Nature Structural
Biol. 1999;6(1):56-63. cited by applicant .
Chakraborty, R., et al., "Identification and mutational studies of
conserved amino acids in the outer membrane receptor protein, FepA,
which affect transport but not binding of ferric-enterobactin in
Escherichia coli," BioMetals 2003;16(4):507-518. cited by applicant
.
Ferguson, A. D., et al., "Structural Basis of Gating by the Outer
Membrane Transporter FecA," Science 2002;295(5560):1715-1719. cited
by applicant .
Howard, S. P., et al., "In Vivo Synthesis of the Periplasmic Domain
of TonB Inhibits Transport through the FecA and FhuA Iron
Siderophore Transporters of Escherichia coli," J. Bacterial.
2001;183(20):5885-5895. cited by applicant .
Killmann, H., et al., "TonB of Escherichia coli activates FhuA
through interaction with the .beta.-barrel," Microbial.
2002;148(11):3497-3509. cited by applicant .
Ogierman, M., et al., "Interactions between the Outer Membrane
Ferric Citrate Transporter FecA and TonB: Studies of the FecA TonB
Box," J. Bacterial. 2003;185(6):1870-1885. cited by applicant .
International Search Report and Written Opinion of the
International Searching Authority for PCT Patent App. No.
PCT/JP2007/051878 (Apr. 19, 2007). cited by applicant .
Garcia-Herrero, A., et al., "Nuclear magnetic resonance solution
structure of the periplastic signalling domain of the
TonB-dependent ou{acute over (t)}er membrane transporter FecA from
Escherichia coli," Mol. Microbiol. 2005;58(5):1226-1237. cited by
applicant .
Howard, S. P., et al., "In Vivo Synthesis of the Periplastic Domain
of TonB Inhibits Transport through the FecA and FhuA Iron
Siderophore Transporters of Escherichia coli," J. Bacteriol.
2001;183(20):5885-5895. cited by applicant .
Murphy, C. K., et al., "Surface Topology of the Escherichia coli
K-12 Ferric Enterobactin Receptor," J. Bacteriol.
1990;172(5):2736-2746. cited by applicant .
Ogierman, M., et al., "Interactions between the Outer Membrane
Ferric Citrate Transporter FecA and TonB: Studies of the FecA TonB
Box," J. Bacteriol. 2003;185(6):1870-1885. cited by applicant .
International Preliminary Report on Patentability for PCT Patent
App. No. PCT/JP2007/051878 (Aug. 14, 2008). cited by
applicant.
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Primary Examiner: Navarro; Albert
Attorney, Agent or Firm: Cermak; Shelly Guest Cermak
Nakajima LLP
Parent Case Text
This application is a continuation under 35 U.S.C. .sctn.120 of
PCT/JP2007/051878, filed Jan. 30, 2007, and claims priority under
35 U.S.C. .sctn.119 to Japanese Patent Application No. 2006-020563,
filed on Jan. 30, 2006, and Japanese Patent Application No.
2006-243282, filed Sep. 7, 2006, all of which are incorporated by
reference. The Sequence Listing filed electronically herewith is
also hereby incorporated by reference in its entirety (File Name:
US-321_Seq_List_Copy.sub.--1; File Size: 72 KB; Date Created: Jul.
25, 2008).
Claims
The invention claimed is:
1. A method of producing an L-amino acid comprising culturing an
L-amino acid-producing bacterium belonging to the
Enterobacteriaceae family in a medium, and collecting the L-amino
acid from the medium or said bacterium, wherein said bacterium has
been modified to enhance the expression of a gene encoding a
protein of the tonB system, wherein said gene is tonB, wherein said
L-amino acid is selected from the group consisting of L-lysine and
L-threonine, wherein said tonB gene encodes a protein comprising an
amino acid sequence which is at least 95% identical to the entire
sequence of SEQ ID NO: 2.
2. The method according to claim 1, wherein the expression is
enhanced by a method selected from the group consisting of: a)
increasing the copy number of said gene, b) modifying an expression
regulatory sequence of said gene, and c) combinations thereof.
3. The method according to claim 1, wherein said tonB encodes a
protein selected from the group consisting of: a) a protein
comprising the amino acid sequence of SEQ ID NO: 2, and b) a
protein comprising the amino acid sequence of SEQ ID NO: 2, wherein
said sequence includes substitutions, deletions, insertions, or
additions of one to 10 amino acids and wherein said protein is able
to regulate the activity of the iron transporter.
4. The method according to claim 1, wherein said tonB is selected
from the group consisting of: a) a DNA comprising the nucleotide
sequence of SEQ ID NO: 1, and b) a DNA that hybridizes with a
nucleotide sequence which is complementary to the nucleotide
sequence of SEQ ID NO: 1 under stringent conditions comprising
washing at a salt concentration of 0.1.times.SSC, 0.1% SDS, at
60.degree. C., and said DNA encodes a protein that is able to
regulate the activity of the iron transporter.
5. The method according to claim 1, wherein said bacterium belongs
to the genus Escherichia, Pantoea, or Enterobacter.
6. The method according to claim 1, wherein said bacterium is
Escherichia coli.
Description
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates to a method of producing an L-amino
acid using a bacterium, and more particularly, to a method of
producing an L-amino acid such as L-lysine, L-threonine, and
L-glutamic acid. L-lysine and L-threonine are useful as additives
in animal feeds, components of health food, amino acid infusions,
and the like. L-glutamic acid is useful as a food seasoning.
2. Brief Description of the Related Art
L-amino acids have been industrially produced by fermentation using
bacteria belonging to the genus Brevibacterium, Corynebacterium,
Escherichia, or the like. Methods of producing L-lysine are
described in EP 0643135 B, EP 0733712 B, EP 1477565 A, EP 0796912
A, EP 0837134 A, WO 01/53459, EP 1170376 A, and WO 2005/010175. In
these methods, bacterial strains are used which are isolated from
nature or artificial mutants thereof, as well as bacterial strains
which have been modified to enhance the activity of an L-amino acid
biosynthetic enzyme by recombinant DNA techniques.
Methods are known for improving L-amino acid-producing ability, and
include modifying the uptake or export of L-amino acids in and out
of cells. A known method of enhancing L-amino acid export is to
produce L-lysine (WO 97/23597) or L-arginine (US 2003-0113899)
using a bacterial strain belonging to the genus Corynebacterium
which has been modified so that expression of an
L-lysine/L-arginine export gene (LysE) is enhanced. In addition,
methods have been reported of producing an L-amino acid using a
bacterium belonging to the Enterobacteriaceae family which has been
modified so that expression is enhanced of the rhtA gene, rhtB
gene, and rhtC gene (EP 1013765 A), yfiK gene or yahN gene (EP
1016710 A), ybjE gene (WO 2005/073390), or yhfk gene (WO
2005/085419). Each of these genes have been suggested to be
involved in L-amino acid export.
It is also known that enhancing the expression of an uptake gene
for a sugar improves the L-amino acid-producing ability. This is
because sugars typically function as a substrate during
fermentation. Examples of such methods include producing an L-amino
acid using an Escherichia bacterium modified to have enhanced
expression of the ptsG gene (WO 03/04670), ptsH gene, ptsI gene, or
crr gene (WO 03/04674).
The fepA gene and the fecA gene each encode a membrane protein
which is known as an iron transporter, while the tonB gene encodes
a protein that regulates the activity of the iron transporter (J.
Bacteriol. 1990; 172(5): 2736-46, J. Bacteriol, 2003, vol. 185, No.
6, p 1870-1885, Mol. Microbiol. 2005; 58(5): 1226-1237, J.
Bacteriol 2001, vol. 183, No. 20, p 5885-5895). However, there have
been no reports that enhancing the activities of these gene
products can be effective for L-amino acid production.
SUMMARY OF THE INVENTION
An object of the present invention is to provide a bacterium which
is capable of effectively producing an L-amino acid and a method of
effectively producing an L-amino acid using the bacterium.
The inventors of the present invention have made extensive studies
to solve the above-mentioned object. As a result, they have found
that production of an L-amino acid is improved by amplifying each
of the genes encoding proteins involved in the tonB system in an
L-amino acid producing bacterium, and thus have completed the
present invention. That is, the present invention is as
follows.
It is an object of the present invention to provide a L-amino
acid-producing bacterium belonging to the Enterobacteriaceae family
which has been modified to enhance the expression of the gene
encoding a protein of the tonB system, and wherein said gene is
selected from the group consisting of the tonB gene, fepA gene,
fecA gene, and combinations thereof.
It is another object of the present invention to provide the
bacterium as described above, wherein the expression is enhanced by
increasing the copy number of said gene(s) or by modifying an
expression regulatory sequence of said gene.
It is another object of the present invention to provide the
bacterium as described above, wherein said tonB gene encodes a
protein having the amino acid sequence of SEQ ID NO: 2 or a protein
having an amino acid sequence of SEQ ID NO: 2, wherein said
sequence includes substitutions, deletions, insertions, or
additions of one or several amino acids and wherein said protein
regulates the activity of the iron transporter.
It is another object of the present invention to provide the
bacterium as described above, wherein said fepA gene encodes a
protein having the amino acid sequence of SEQ ID NO: 4 or a protein
having an amino acid sequence of SEQ ID NO: 4, wherein said
sequence includes substitutions, deletions, insertions, or
additions of one or several amino acids, and wherein said protein
has iron transporter activity.
It is another object of the present invention to provide the
bacterium as described above, wherein said fecA gene encodes a
protein having the amino acid sequence of SEQ ID NO: 10 or a
protein having an amino acid sequence of SEQ ID NO: 10, wherein
said sequence includes substitutions, deletions, insertions, or
additions of one or several amino acids, wherein said protein has
iron transporter activity.
It is another object of the present invention to provide the
bacterium as described above, wherein said tonB gene is selected
from the group consisting of: (a) a DNA comprising the nucleotide
sequence of SEQ ID NO: 1; and (b) a DNA that hybridizes with a
nucleotide sequence which is complementary to the nucleotide
sequence of SEQ ID NO: 1 or with a probe that is prepared from the
nucleotide sequence under stringent conditions, and wherein said
DNA encodes a protein that is able to regulate the activity of the
iron transporter.
It is another object of the present invention to provide the
bacterium as described above, wherein said fepA gene is selected
from the group consisting of: (c) a DNA comprising the nucleotide
sequence of SEQ ID NO: 3; and (d) a DNA that hybridizes with a
nucleotide sequence which is complementary to the nucleotide
sequence of SEQ ID NO: 3 or a probe that is prepared from the
nucleotide sequence under stringent conditions, and wherein said
DNA encodes a protein that has iron transporter activity.
It is another object of the present invention to provide the
bacterium as described above, wherein the fecA gene is selected
from the group consisting of: (e) a DNA comprising the nucleotide
sequence of SEQ ID NO: 9; and (f) a DNA that hybridizes with a
nucleotide sequence which is complementary to the nucleotide
sequence of SEQ ID NO: 9 or a probe that is prepared from the
nucleotide sequence under stringent conditions, and wherein said
DNA encodes a protein that has iron transporter activity.
It is another object of the present invention to provide the
bacterium as described above, wherein the L-amino acid is selected
from the group consisting of L-lysine, L-arginine, L-histidine,
L-isoleucine, L-valine, L-leucine, L-threonine, L-phenylalanine,
L-tyrosine, L-tryptophan, L-cysteine, L-glutamic acid, and
combinations thereof.
It is another object of the present invention to provide the
bacterium as described above, wherein said bacterium belongs to the
genus Escherichia, Pantoea, or Enterobacter.
It is another object of the present invention to provide a method
of producing an L-amino acid, comprising culturing the bacterium as
described above in a medium to produce and accumulate an L-amino
acid in the medium or bacterial cells, and collecting the L-amino
acid from the medium or bacterial cells.
It is another object of the present invention to provide the method
as described above, wherein said L-amino acid is selected from the
group consisting of L-lysine, L-arginine, L-histidine,
L-isoleucine, L-valine, L-leucine, L-threonine, L-phenylalanine,
L-tyrosine, L-tryptophan, L-cysteine, L-glutamic acid, and
combinations thereof.
DESCRIPTION OF THE PREFERRED EMBODIMENTS
Hereinafter, the present invention will be described in detail.
<1> Bacterium of the Present Invention
The bacterium of the present invention belongs to the
Enterobacteriaceae family, and has an L-amino acid-producing
ability, and is modified so that the activity of an iron
transporter is enhanced by enhancing the expression of a gene
encoding a protein of the tonB system. Herein, the term "L-amino
acid-producing ability" refers to the ability to produce and
accumulate an L-amino acid in a medium at a collectable level when
the bacterium of the present invention is cultured in the medium.
The bacterium of the present invention may be able to produce a
plurality of L-amino acids. The L-amino acid-producing ability may
be native to the bacterium, or may be obtained by modifying the
bacterium to impart the L-amino acid-producing ability by mutation
or a recombinant DNA technique.
The kind of L-amino acid is not particularly limited, and examples
thereof include the basic L-amino acids such as L-lysine,
L-ornithine, L-arginine, L-histidine and L-citrulline; the
aliphatic L-amino acids such as L-isoleucine, L-alanine, L-valine,
L-leucine, and L-glycine; the hydroxy monoaminocarboxylic acids
such as L-threonine and L-serine; the cyclic L-amino acids such as
L-proline; the aromatic L-amino acids such as L-phenylalanine,
L-tyrosine, and L-tryptophan; the sulfur-containing L-amino acids
such as L-cysteine, L-cystine, and L-methionine; and the acidic
L-amino acids such as L-glutamic acid, L-aspartic acid,
L-glutamine, and L-asparagine. The bacterium of the present
invention may be able to produce two or more kinds of amino
acids.
<1-1> Imparting L-Amino Acid-Producing Ability
Hereinafter, methods of imparting the L-amino acid-producing
ability will be described, as well as examples of the bacteria to
which an L-amino acid-producing ability can be imparted. However,
the bacterium is not limited thereto, as long as it has an L-amino
acid-producing ability.
Bacteria belonging to the Enterobacteriaceae family, including
those belonging to the genus Escherichia or Pantoea, can be used as
the parent strain from which to derive the bacterium of the present
invention. Other examples of bacteria belonging to the
Enterobacteriaceae family include .gamma.-Proteobacteria such as
Enterobacter, Klebsiella, Serratia, Erwinia, Salmonella, and
Morganella. Escherichia bacteria reported in Neidhardt et al.
((Backmann, B. J. 1996. Derivations and Genotypes of some mutant
derivatives of Escherichia coli K-12, p. 2460-2488. Table 1. In F.
D. Neidhardt (ed.), Escherichia coli and Salmonella Cellular and
Molecular Biology/Second Edition, American Society for Microbiology
Press, Washington, D.C.), such as Escherichia coli can be utilized.
Examples of a wild-type strain of Escherichia coli include the K-12
strain or derivatives thereof, Escherichia coli MG1655 strain (ATCC
No. 47076), and W3110 strain (ATCC No. 27325). These strains are
available from the American Type Culture Collection (ATCC)
(Address: P.O. Box 1549, Manassas, Va. 20108, 1, United States of
America).
Examples of Enterobacter bacteria include Enterobacter agglomerans
and Enterobacter aerogenes, and an example of Pantoea bacteria is
Pantoea ananatis. Recently, Enterobacter agglomerans was
reclassified in some cases as Pantoea agglomerans, Pantoea
ananatis, Pantoea stewartii, or the like, based on an analysis of
the nucleotide sequence of 16S rRNA. Therefore, bacteria of the
present invention may belong to either the genus Enterobacter or
the genus Pantoea, as long as they are classified in the
Enterobacteriaceae family. When Pantoea ananatis is bred using
genetic engineering techniques, Pantoea ananatis AJ13355 strain
(FERM BP-6614), AJ13356 strain (FERM BP-6615), AJ13601 strain (FERM
BP-7207), derivatives thereof, and the like, may be used. These
strains were identified and deposited as Enterobacter agglomerans
when they were isolated, but as described above, these strains have
been reclassified as Pantoea ananatis based on an analysis of the
nucleotide sequence of 16S rRNA.
The L-amino acid-producing ability can be imparted to a parent
strain as described above, as follows.
In order to impart the L-amino acid-producing ability, methods may
be used which are used in conventional breeding of Escherichia
bacteria or the like, such as by acquiring nutrient-auxotrophic
mutant strains, analogue resistant strains, or metabolic regulation
mutant strains, or by creating recombinant strains having enhanced
expression of L-amino acid biosynthetic enzymes (Amino Acid
Fermentation, Japan Scientific Societies Press, first edition
publication: May 30, 1986, p. 77 to 100). In the present invention,
properties such as nutrient-auxotrophy, analogue-resistance, and
metabolic regulation may be imparted alone or in combination with
imparting the L-amino acid-producing ability. Furthermore,
expression of one or more L-amino acid biosynthetic enzymes may be
enhanced. Furthermore, imparting of such properties as
nutrient-auxotrophy, analogue-resistance and metabolic regulation
mutation may be combined with enhancing the expression of the
L-amino acid biosynthetic enzymes.
Nutrient-auxotrophic mutant strains, L-amino acid-analogue
resistant strains, and metabolic regulation mutant strains that
have an L-amino acid-producing ability can be obtained as follows.
A parent strain or a wild-type strain is subjected to a typical
mutation treatment, such as irradiation with X-rays or ultraviolet
rays, or by treating with a mutagen, including
N-methyl-N'-nitro-N-nitrosoguanidine (NTG) and
ethylmethanesulfonate (EMS), followed by selection of the strains
that exhibit nutrient-auxotrophy, analogue-resistance, or a
metabolic regulation mutation and have an L-amino acid-producing
ability.
Examples of an L-lysine analogue include oxalysine, lysine
hydroxamate, S-(2-aminoethyl)-L-cysteine (AEC),
.gamma.-methyllysine, .alpha.-chlorocaprolactam, and norleucine.
Examples of an L-arginine analogue include arginine hydroxamate,
homoarginine, D-arginine, and canavanine.
Specific examples of an L-lysine analogue resistant strain or
metabolic regulation mutant strain having an L-lysine-producing
ability include Escherichia coli AJ11442 (FERM BP-1543, NRRL
B-12185; JP 56-18596 A and U.S. Pat. No. 4,346,170) and Escherichia
coli VL611 (JP 2000-189180 A). The WC196 strain (WO 96/17930) may
be used as an L-lysine producing strain of Escherichia coli. The
WC1-96 strain was obtained by imparting AEC
(S-(2-aminoethyl)-cysteine)-resistance to the W3110 strain, which
was derived from Escherichia coli K-12 strain. The WC196 strain was
named Escherichia coli AJ13069 and deposited at the National
Institute of Bioscience and Human-Technology, Agency of Industrial
Science and Technology (currently, International Patent Organism
Depositary, National Institute of Advanced Industrial Science and
Technology, Central 6, 1-1-1 Higashi, Tsukuba, Ibaraki 305-8566,
Japan) on Dec. 6, 1994 and given an accession number of FERM
P-14690, and the deposit was then converted to an international
deposit under the provisions of Budapest Treaty on Sep. 29, 1995
and given an accession number of FERM BP-5252.
An L-amino acid-producing ability can also be imparted by enhancing
the expression of a gene encoding an L-amino acid biosynthetic
enzyme.
For example, as described below, an L-lysine-producing ability may
be imparted by enhancing the activities of dihydrodipicolinate
synthase and aspartokinase. That is, a gene fragment encoding
dihydrodipicolinate synthase and a gene fragment encoding
aspartokinase are ligated to a vector which functions in the host
bacterium. The vector is preferably a multi-copy vector, and is
used to transform the host bacterium. The transformation results in
increased copy numbers of the gene encoding dihydrodipicolinate
synthase and the gene encoding aspartokinase in the host cell,
thereby enhancing the activities of these enzymes. Hereinafter,
dihydrodipicolinate synthase, aspartokinase, and aspartokinase III
are abbreviated as DDPS, AK, and AKIII, respectively.
The genes encoding DDPS and AK are not particularly limited as long
as the DDPS and AK activities are expressed in the host bacterium,
and examples thereof include the genes of Escherichia coli,
Methylophilus methylotrophus, Corynebacterium glutamicum, and the
like. The nucleotide sequences of the DDPS gene derived from an
Escherichia bacterium (dapA, Richaud, F. et al. J. Bacteriol., 297
(1986)) and the AKIII gene derived from an Escherichia bacterium
(lysC, Cassan, M., Parsot, C., Cohen, G. N. and Patte, J. C., J.
Biol. Chem., 261, 1052 (1986)) have been identified, so these genes
can be obtained by PCR using primers synthesized based on their
nucleotide sequences and the chromosomal DNA of Escherichia coli
K-12, for example, as a template. Hereinafter, dapA and lysC
derived from Escherichia coli will be exemplary, but the genes
encoding DDPS and AK are not limited thereto.
It is known that the wild-type DDPS derived from Escherichia coli
is regulated by feedback inhibition by L-lysine, while the
wild-type AKIII derived from Escherichia coli is regulated by
suppression and feedback inhibition by L-lysine. Therefore, when
using dapA and lysC, mutated forms of these genes are preferable so
that the genes are not subject to feedback inhibition. However, the
DDPS and AK of the present invention are not necessarily these
mutants since the DDPS derived from Corynebacterium bacterium is
not subject to feedback inhibition.
An example of a DNA encoding mutant DDPS which is not subject to
feedback inhibition by L-lysine includes a DNA encoding DDPS which
has an amino acid sequence in which the histidine at position 118
is substituted with tyrosine. Meanwhile, an example of a DNA
encoding mutant AKIII which is not subject to feedback inhibition
by L-lysine includes a DNA encoding an AKIII having an amino acid
sequence in which the threonine at position 352, the glycine at
position 323, and the methionine at position 318 are replaced with
isoleucine, asparagine and isoleucine, respectively (U.S. Pat. No.
5,661,012 and U.S. Pat. No. 6,040,160). Such mutant DNAs can be
obtained by site-specific mutation using PCR or the like.
Enhancing expression of the L-lysine biosynthetic genes as
described above can be attained by transformation or homologous
recombination using a plasmid or the like, in the same way as the
tonB gene, fepA gene, and fecA gene described below.
Wide host-range plasmids RSFD80, pCAB1, and pCABD2 contain a mutant
dapA gene encoding a mutant DDPS and a mutant lysC gene encoding a
mutant AKIII (U.S. Pat. No. 6,040,160). Escherichia coli JM109
strain transformed with RSFD80 was named AJ12396 (U.S. Pat. No.
6,040,160), and the strain was deposited at the National Institute
of Bioscience and Human-Technology, Agency of Industrial Science
and Technology, Ministry of International Trade and Industry
(currently, International Patent Organism Depositary, National
Institute of Advanced Industrial Science and Technology) on Oct.
28, 1993 and given an accession number of FERM P-13936, and the
deposit was then converted to an international deposit under the
provisions of Budapest Treaty on Nov. 1, 1994 and given an
accession number of FERM BP-4859. RSFD80 can be obtained from
AJ12396 strain by a conventional method.
An L-lysine-producing ability can also be imparted by enhancing
expression of genes encoding enzymes, other than DDPS and AK, which
are involved in biosynthesis of L-lysine. Examples of such enzymes
include proteins of the diaminopimelate pathway such as
dihydrodipicolinate reductase (dapB: hereinafter, the words in
parentheses are the gene names) (WO01/53459), diaminopimelate
decarboxylase (lysA), diaminopimelate dehydrogenase (ddh)
(WO96/40934), phosphoenolpyruvate carboxylase (pepC) (JP 60-87788
A), aspartate aminotransferase (aspC) (JP 06-102028 B),
diaminopimelate epimerase gene (dapF) (JP 2003-135066), aspartate
semialdehyde dehydrogenase (asd) (WO 00/61723),
tetrahydrodipicolinate succinylase (dapD), and
succinyl-diaminopimelate deacylase (dapE). Further examples are
proteins of the aminoadipic acid pathway such as homoaconitate
hydratase (JP 2000-157276 A). The documents indicated in
parentheses disclose L-lysine-producing strains having enhanced
expression of a gene encoding each enzyme. Enhancing expression of
a gene encoding each enzyme may be combined with enhancing
expression of the DDPS and AK genes.
Expression of genes other than L-lysine biosynthetic genes may also
be enhanced, and such genes include those encoding enzymes involved
in sugar uptake, sugar metabolism (glycolytic pathway), the TCA
cycle, the pentose phosphate cycle, complementary pathway, and
energy metabolism. Moreover, the expression may also be enhanced of
genes that impart amino acid-resistance to a host bacterium, genes
encoding amino acid-export enzymes, and genes encoding enzymes
involved in uptake of by-products. Enhancing the expression of
these genes is useful for the production of all kinds of L-amino
acids.
Genes involved in sugar metabolism include genes encoding enzymes
in the glycolytic pathway or enzymes involved in sugar uptake.
Examples thereof include the glucose-6-phosphate isomerase gene
(pgi; WO 01/02542), phosphoenolpyruvate synthase gene (pps; EP
877090 A), phosphoglucomutase gene (pgm; WO 03/04598), fructose
bisphosphate aldolase gene (fbp; WO 03/04664), pyruvate kinase gene
(pykF; WO 03/008609), transaldolase gene (talB; WO 03/008611),
fumarase gene (fum; WO 01/02545), phosphoenolpyruvate synthase gene
(pps; EP 877090 A), non-PTS sucrose uptake gene (csc; EP 149911 A),
sucrose-assimilating gene (scrAB operon; WO 90/04636), PTS glucose
uptake gene (ptsG, ptsH, ptsI, crr; WO 03/04670, WO 03/04674, and
EP 1254957 A), galactose-proton symporter gene (galP;
US2004-214294), D-xylose permease gene (xylE;
WO2006/043730.quadrature. and a gene involved in maltose transport
(malK; EP 1254957).
Examples of genes encoding the TCA cycle enzymes include the
citrate synthase gene (gltA; WO 03/008607), isocitrate
dehydrogenase gene (icd; WO 03/008607), 2-ketoglutarate
dehydrogenase gene (sucAB; WO 03/008614), succinate dehydrogenase
gene (sdh; WO 01/02544), and glutamate dehydrogenase gene (gdh;
WO00/53726).
Examples of genes encoding the pentose phosphate cycle enzymes
include the glucose-6-phosphate dehydrogenase gene (zwf; WO
03/008607) and ribose-5-phosphate isomerase gene (rpiB; WO
03/008607).
Examples of genes encoding the anaplerotic pathway include the
phosphoenolpyruvate carboxylase gene (pepC; U.S. Pat. No.
5,876,983), pyruvate carboxylase gene (pyc; EP 1092776), malate
dehydrogenase gene (mdh; WO 01/02546), and phosphoenolpyruvate
carboxykinase gene (pckA; WO 04/090125).
Examples of genes encoding enzymes involved in energy metabolism
include the transhydrogenase gene (pntAB; U.S. Pat. No. 5,830,716)
and cytochromoe bo type oxidase gene (cyoB; EP 1070376).
Examples of genes that impart L-amino acid-resistance include the
rhtB gene (U.S. Pat. No. 6,887,691), rhtC gene (EP 1013765), yedA
gene (EP 1449917), yddG gene (EP 1449918), ygaZH gene (EP 1239041),
yahN, yfiK, and yeaS genes (EP 1016710), rhtA gene (Res Microbiol.
2003 March; 154(2): 123-35), and ybjE gene (WO 2005/073390).
Furthermore, in the bacterium of the present invention, the
activity of an enzyme that catalyzes a reaction which branches off
from the L-lysine biosynthetic pathway and produces a compound
other than L-lysine may be decreased or may be made deficient.
Examples of such an enzyme include homoserine dehydrogenase, lysine
decarboxylase, and malic enzyme, and strains in which the
activities of such enzymes are decreased or deficient are described
in WO 95/23864, WO 96/17930, WO 2006/038695, WO 2005/010175, and
the like. In Escherichia coli, lysine decarboxylases are encoded by
the cadA gene (Genbank Accession No. NP.sub.--418555, SEQ ID NO:
17) and ldcC gene (Genbank Accession No. NP.sub.--414728, SEQ ID
NO: 11) (WO 96/17930), so these genes may be disrupted to enhance
L-lysine-producing ability. DNA molecules homologous to the cadA
gene and ldcC gene may be used as long as they can cause homologous
recombination with the cadA gene and ldcC gene on the chromosome of
the host bacterium. For example, a DNA molecule homologous to the
cadA gene may hybridize to the complementary strand of SEQ ID NO:
17 under stringent conditions, and a DNA molecule homologous to the
ldcC gene may hybridize to the complementary strand of SEQ ID NO:
11 under stringent conditions.
Activities of these enzymes can be decreased or eliminated by
introducing a mutation into the genes encoding the enzymes on the
chromosome using a known mutation treatment, to thereby decrease or
eliminate the activities of the enzymes in the cell. For example,
decreasing or eliminating the activities of the enzymes can be
attained by disrupting the genes encoding the enzymes on the
chromosome by gene recombination or by modifying an expression
regulatory sequence such as the promoter or Shine-Dalgarno (SD)
sequence. In addition, this can also be attained by introducing an
amino acid substitution (missense mutation) to the region encoding
the enzymes on the chromosome, introducing a stop codon (nonsense
mutation), introducing a frameshift mutation that adds or deletes
one or two nucleotides, or deleting part of the gene (Journal of
biological Chemistry 272: 8611-8617 (1997). Also, the activities of
the enzymes can also be decreased or eliminated by constructing a
mutant gene which has a deletion in the coding region, and then
replacing the normal gene on the chromosome with the mutant gene by
homologous recombination, or introducing the mutant gene using a
transposon or an IS factor.
For example, the following gene recombination method can be used to
introduce a mutation that decreases or eliminates the activities of
the above-mentioned enzymes. The mutant gene is prepared by
modifying a partial sequence of a target gene so that it does not
encode an properly-functioning enzyme. Then, a bacterium belonging
to the Enterobacteriaceae family is transformed with a DNA
containing the mutant, resulting in recombination of a gene on the
bacterial chromosome with the mutant gene, thereby substituting the
target gene on the chromosome with the mutant gene. Examples of
this type of gene substitution using homologous recombination using
a linear DNA called "Red-driven integration" (Datsenko, K. A, and
Wanner, B. L. Proc. Natl. Acad. Sci. USA. 97: 6640-6645 (2000), a
combination of Red-driven integration and a cleavage system derived
from .lamda. phage (Cho, E. H., Gumport, R. I., Gardner, J. F. J.
Bacteriol. 184: 5200-5203 (2002)) (WO 2005/010175), using a plasmid
containing a temperature-sensitive replication origin (Datsenko, K.
A, and Wanner, B. L. Proc. Natl. Acad. Sci. USA. 97: 6640-6645
(2000); U.S. Pat. No. 6,303,383; JP 05-007491 A), and the like.
Meanwhile, site-specific mutation by gene substitution using
homologous recombination can also be performed by using a plasmid
which is not able to replicate in the host cell.
The above-described methods for enhancing the expression of the
L-lysine biosynthetic enzymes' genes and for decreasing the
activities of enzymes can also be applied to genes encoding other
L-amino acid synthetic enzymes. In this way, the ability to produce
another L-amino acid can be imparted to a bacterium of the
Enterobacteriaceae family.
Hereinafter, a bacterium to which an ability to produce an L-amino
acid other than L-lysine is imparted will be exemplified.
L-Threonine-Producing Bacteria
Examples of parent strains for deriving the L-threonine-producing
bacteria of the present invention include, but are not limited to,
strains belonging to the genus Escherichia, such as E. coli
TDH-6/pVIC40 (VKPM B-3996) (U.S. Pat. No. 5,175,107, U.S. Pat. No.
5,705,371), E. coli 472T23/pYN7 (ATCC 98081) (U.S. Pat. No.
5,631,157), E. coli NRRL-21593 (U.S. Pat. No. 5,939,307), E. coli
FERM BP-3756 (U.S. Pat. No. 5,474,918), E. coli FERM BP-3519 and
FERM BP-3520 (U.S. Pat. No. 5,376,538), E. coli MG442 (Gusyatiner
et al., Genetika (in Russian), 14, 947-956 (1978)), E. coli VL643
and VL2055 (EP 1149911 A), and the like.
The TDH-6 strain is deficient in the thrc gene, as well as being
sucrose-assimilative, and the ilvA gene has a leaky mutation. This
strain also has a mutation in the rhtA gene, which imparts
resistance to high concentrations of threonine or homoserine. The
B-3996 strain contains pVIC40, which was obtained by inserting the
thrA*BC operon which includes a mutant thrA gene into a
RSF110-derived vector. This mutant thrA gene encodes aspartokinase
homoserine dehydrogenase I which is substantially desensitized to
feedback inhibition by threonine. The B-3996 strain was deposited
on Nov. 19, 1987 in the All-Union Scientific Center of Antibiotics
(Nagatinskaya Street 3-A, 117105 Moscow, Russian Federation) under
the accession number RIA 1867. This strain was also deposited in
the Russian National Collection of Industrial Microorganisms (VKPM)
(Russia, 117545 Moscow 1, Dorozhny proezd. 1) on Apr. 7, 1987 under
the accession number B-3996.
E. coli VKPM B-5318 (EP 0593792B) may also be used to derive the
L-threonine-producing bacteria of the present invention. The B-5318
strain is prototrophic with regard to isoleucine, and a
temperature-sensitive lambda-phage C1 repressor and PR promoter
replaces the regulatory region of the threonine operon in plasmid
pVIC40. The VKPM B-5318 strain was deposited in the Russian
National Collection of Industrial Microorganisms (VKPM) (Russia,
117545 Moscow 1, Dorozhny proezd. 1) on May 3, 1990 under accession
number of VKPM B-5318.
Preferably, the bacterium of the present invention is additionally
modified to enhance expression of one or more of the following
genes: the mutant thrA gene which codes for aspartokinase
homoserine dehydrogenase I resistant to feed back inhibition by
threonine; the thrB gene which codes for homoserine kinase; the
thrc gene which codes for threonine synthase; the rhtA gene which
codes for a putative transmembrane protein; the asd gene which
codes for aspartate-.alpha.-semialdehyde dehydrogenase; and the
aspC gene which codes for aspartate aminotransferase (aspartate
transaminase).
The sequence of the thrA gene of Escherichia coli which encodes
aspartokinase homoserine dehydrogenase I has been elucidated
(nucleotide positions 337 to 2799, GenBank accession
NC.sub.--000913.2, gi: 49175990). The thrA gene is located between
the thrL and thrB genes on the chromosome of E. coli K-12. The
nucleotide sequence of the thrB gene of Escherichia coli which
encodes homoserine kinase has been elucidated (nucleotide positions
2801 to 3733, GenBank accession NC.sub.--000913.2, gi: 49175990).
The thrB gene is located between the thrA and thrC genes on the
chromosome of E. coli K-12. The nucleotide sequence of the thrC
gene of Escherichia coli which encodes threonine synthase has been
elucidated (nucleotide positions 3734 to 5020, GenBank accession
NC.sub.--000913.2, gi: 49175990). The thrC gene is located between
the thrB gene and the yaax open reading frame on the chromosome of
E. coli K-12. All three genes function together as a single
threonine operon. To enhance the expression of the threonine
operon, the attenuator region which affects the transcription can
be removed from the operon (WO2005/049808, WO2003/097839).
The mutated thrA gene which encodes feedback-resistant
aspartokinase homoserine dehydrogenase I, as well as the thrB and
thrc genes can be obtained as one operon from the well-known
plasmid pVIC40. This plasmid is present in the threonine producing
E. coli strain VKPM B-3996, and is described in detail in U.S. Pat.
No. 5,705,371.
The rhtA gene is at 18 min on the E. coli chromosome close to the
glnHPQ operon, which encodes components of the glutamine transport
system. The rhtA gene is identical to ORF1 (ybiF gene, nucleotide
positions 764 to 1651, GenBank accession number AAA218541,
gi:440181) and is located between the pexB and ompX genes. The
sequence expressing a protein encoded by the ORF1 has been
designated the rhtA gene (rht: resistance to homoserine and
threonine). Also, the rhtA23 mutation is an A-for-G substitution at
position-1 with respect to the ATG start codon (ABSTRACTS of the
17th International Congress of Biochemistry and Molecular Biology
in conjugation with Annual Meeting of the American Society for
Biochemistry and Molecular Biology, San Francisco, Calif. Aug.
24-29, 1997, abstract No. 457, EP 1013765 A).
The nucleotide sequence of the asd gene of E. coli has already been
elucidated (nucleotide positions 3572511 to 3571408, GenBank
accession NC.sub.--000913.1, gi:16131307), and can be obtained by
PCR (polymerase chain reaction; refer to White, T. J. et al.,
Trends Genet., 5, 185 (1989)) by utilizing primers based on the
nucleotide sequence of the gene. The asd genes from other
microorganisms can be obtained in a similar manner.
Also, the nucleotide sequence of the aspC gene of E. coli has
already been elucidated (nucleotide positions 983742 to 984932,
GenBank accession NC.sub.--000913.1, gi:16128895), and can be
obtained by PCR. The aspC genes from other microorganisms can be
obtained in a similar manner.
L-Cysteine-Producing Bacteria
Examples of parent strains for deriving L-cysteine-producing
bacteria of the present invention include, but are not limited to,
strains belonging to the genus Escherichia, such as E. coli JM15
which has been transformed with different cysE alleles coding for
feedback-resistant serine acetyltransferases (U.S. Pat. No.
6,218,168, Russian patent application 2003121601), E. coli W3110
which over-expresses genes which encode proteins suitable for
secreting toxic substances (U.S. Pat. No. 5,972,663), E. coli
strains with decreased cysteine desulfohydrase activity
(JP11155571A2); E. coli W3110 with increased activity of a positive
transcriptional regulator for the cysteine regulon encoded by the
cysB gene (WO0127307A1), and the like.
L-Leucine-Producing Bacteria
Examples of parent strains for deriving L-leucine-producing
bacteria of the present invention include, but are not limited to,
strains belonging to the genus Escherichia, such as E. coli strains
resistant to leucine (for example, the strain 57 (VKPM B-7386, U.S.
Pat. No. 6,124,121)) or leucine analogs including
P3-2-thienylalanine, 3-hydroxyleucine, 4-azaleucine,
5,5,5-trifluoroleucine (JP 62-34397 B and JP 8-70879 A); E. coli
strains obtained by the genetic engineering method described in
WO96/06926; E. coli H-9068 (JP 8-70879 A), and the like.
The bacterium of the present invention may be improved by enhancing
the expression of one or more genes involved in L-leucine
biosynthesis. Examples of these genes include those of the leuABCD
operon, which preferably include a leuA gene which has been mutated
so that it encodes isopropylmalate synthase which is resistant to
feedback inhibition by L-leucine (U.S. Pat. No. 6,403,342). In
addition, the bacterium of the present invention may be improved by
enhancing the expression of one or more genes coding for proteins
which excrete L-amino acids from the bacterial cell. Examples of
such genes include the b2682 and b2683 genes (ygaZH genes) (EP
1239041 A2).
L-Histidine-Producing Bacteria
Examples of parent strains for deriving L-histidine-producing
bacteria of the present invention include, but are not limited to,
strains belonging to the genus Escherichia, such as E. coli strain
24 (VKPM B-5945, RU2003677); E. coli strain 80 (VKPM B-7270,
RU2119536); E. coli NRRL B-12116-B12121 (U.S. Pat. No. 4,388,405);
E. coli H-9342 (FERM BP-6675) and H-9343 (FERM BP-6676) (U.S. Pat.
No. 6,344,347); E. coli H-9341 (FERM BP-6674) (EP1085087); E. coli
A180/pFM201 (U.S. Pat. No. 6,258,554) and the like.
Examples of parent strains for deriving L-histidine-producing
bacteria of the present invention also include strains in which
expression of one or more genes encoding an L-histidine
biosynthetic enzyme are enhanced. Examples of these
L-histidine-biosynthetic enzymes include ATP
phosphoribosyltransferase (hisG), phosphoribosyl AMP cyclohydrolase
(hisI), phosphoribosyl-ATP pyrophosphohydrolase (hisIE),
phosphoribosylformimino-5-aminoimidazole carboxamide ribotide
isomerase (hisA), amidotransferase (hisH), histidinol phosphate
aminotransferase (hisC), histidinol phosphatase (hisB), histidinol
dehydrogenase (hisD), and so forth.
It is known that the genes encoding the L-histidine biosynthetic
enzyme (hisG, hisBHAFI) are inhibited by L-histidine, and therefore
the L-histidine-producing ability can also be efficiently enhanced
by introducing a mutation which induces resistance to the feedback
inhibition into ATP phosphoribosyltransferase (hisG) (Russian
Patent Nos. 2003677 and 2119536).
Specific examples of strains having an L-histidine-producing
ability include E. coli FERM-P 5038 and 5048 which have been
transformed with a vector carrying a DNA encoding an
L-histidine-biosynthetic enzyme (JP 56-005099 A), E. coli strains
transformed with rht, a gene for an amino acid-export (EP1016710A),
E. coli 80 strain imparted with sulfaguanidine,
DL-1,2,4-triazole-3-alanine, and streptomycin-resistance (VKPM
B-7270, Russian Patent No. 2119536), and so forth.
L-Glutamic Acid-Producing Bacteria
Examples of parent strains for deriving L-glutamic acid-producing
bacteria of the present invention include, but are not limited to,
strains belonging to the genus Escherichia, such as E. coli
VL334thrC.sup.+ (EP 1172433). E. coli VL334 (VKPM B-1641) is
auxotrophic for L-isoleucine and L-threonine and is mutated in the
thrC and ilvA genes (U.S. Pat. No. 4,278,765). A wild-type allele
of the thrc gene was transferred by general transduction using a
bacteriophage P1 grown on the wild-type E. coli strain K12 (VKPM
B-7). As a result, an L-isoleucine auxotrophic strain
VL334thrC.sup.+ (VKPM B-8961) was obtained.
Examples of parent strains for deriving the L-glutamic
acid-producing bacteria of the present invention include, but are
not limited to, strains in which expression of one or more genes
encoding an L-glutamic acid biosynthetic enzyme are enhanced.
Examples of the enzymes involved in L-glutamic acid biosynthesis
include glutamate dehydrogenase (gdhA), glutamine synthetase
(glnA), glutamate synthetase (gltAB), isocitrate dehydrogenase
(icdA), aconitate hydratase (acnA, acnB), citrate synthase (gltA),
phosphoenolpyruvate carboxylase (ppc), pyruvate dehydrogenase
(aceEF, lpdA), pyruvate kinase (pykA, pykF), phosphoenolpyruvate
synthase (ppsA), enolase (eno), phosphoglyceromutase (pgmA),
phosphoglycerate kinase (pgk), glyceraldehyde-3-phophate
dehydrogenase (gapA), triose phosphate isomerase (tpiA), fructose
bisphosphate aldolase (fbp), phosphofructokinase (pfkA, pfkB), and
glucose phosphate isomerase (pgi).
Examples of strains modified so that expression of the citrate
synthetase gene, the phosphoenolpyruvate carboxylase gene, and/or
the glutamate dehydrogenase gene is/are enhanced include those
disclosed in EP1078989A, EP955368A, and EP952221A.
Examples of parent strains for deriving the L-glutamic
acid-producing bacteria of the present invention also include
strains which have decreased or eliminated activity of an enzyme
that catalyzes synthesis of a compound other than L-glutamic acid,
and branches off from the L-glutamic acid biosynthesis pathway.
Examples of such enzymes include isocitrate lyase,
.alpha.-ketoglutarate dehydrogenase, phosphotransacetylase, acetate
kinase, acetohydroxy acid synthase, acetolactate synthase, formate
acetyltransferase, lactate dehydrogenase, and glutamate
decarboxylase. Bacteria belonging to the genus Escherichia
deficient in the .alpha.-ketoglutarate dehydrogenase activity or
having a reduced .alpha.-ketoglutarate dehydrogenase activity and
methods for obtaining them are described in U.S. Pat. Nos.
5,378,616 and 5,573,945.
Specifically, these strains include the following: E. coli
W3110sucA:Kmr E. coli AJ12624 (FERM BP-3853) E. coli AJ12628 (FERM
BP-3854) E. coli AJ12949 (FERM BP-4881)
E. coli W3110sucA:Kmr is obtained by disrupting the
.alpha.-ketoglutarate dehydrogenase gene (hereinafter referred to
as "sucA gene") of E. coli W3110. This strain is completely
deficient in .alpha.-ketoglutarate dehydrogenase.
Other examples of L-glutamic acid-producing bacterium include those
which belong to the genus Escherichia and have resistance to an
aspartic acid antimetabolite. These strains can also be deficient
in .alpha.-ketoglutarate dehydrogenase activity and include, for
example, E. coli AJ13199 (FERM BP-5807) (U.S. Pat. No. 5,908,768),
FERM P-12379, which additionally has a low L-glutamic acid
decomposing ability (U.S. Pat. No. 5,393,671); AJ13138 (FERM
BP-5565) (U.S. Pat. No. 6,110,714), and the like.
Examples of L-glutamic acid-producing bacteria include mutant
strains belonging to the genus Pantoea which are deficient in
.alpha.-ketoglutarate dehydrogenase activity or have a decreased
.alpha.-ketoglutarate dehydrogenase activity, and can be obtained
as described above. Such strains include Pantoea ananatis AJ13356
(U.S. Pat. No. 6,331,419). Pantoea ananatis AJ13356 was deposited
at the National Institute of Bioscience and Human-Technology,
Agency of Industrial Science and Technology, Ministry of
International Trade and Industry (currently, National Institute of
Advanced Industrial Science and Technology, International Patent
Organism Depositary, Central 6, 1-1, Higashi 1-Chome, Tsukuba-shi,
Ibaraki-ken, 305-8566, Japan) on Feb. 19, 1998 under an accession
number of FERM P-16645. It was then converted to an international
deposit under the provisions of Budapest Treaty on Jan. 11, 1999
and received an accession number of FERM BP-6615. Pantoea ananatis
AJ13356 is deficient in .alpha.-ketoglutarate dehydrogenase
activity as a result of the disruption of the .alpha.LKGDH-E1
subunit gene (sucA). The above strain was identified as
Enterobacter agglomerans when it was isolated and deposited as the
Enterobacter agglomerans AJ13356. However, it was recently
re-classified as Pantoea ananatis on the basis of nucleotide
sequencing of 16S rRNA and so forth. Although AJ13356 was deposited
at the aforementioned depository as Enterobacter agglomerans, for
the purposes of this specification, they are described as Pantoea
ananatis.
L-Phenylalanine-Producing Bacteria
Examples of parent strains for deriving L-phenylalanine-producing
bacteria of the present invention include, but are not limited to,
strains belonging to the genus Escherichia, such as E. coli AJ12739
(tyrA:Tn10, tyrR) (VKPM B-8197); E. coli HW1089 (ATCC 55371)
harboring the pheA34 gene (U.S. Pat. No. 5,354,672); E. coli
MWEC101-b (KR8903681); E. coli NRRL B-12141, NRRL B-12145, NRRL
B-12146 and NRRL B-12147 (U.S. Pat. No. 4,407,952). Also, as a
parent strain, E. coli K-12 [W3110 (tyrA)/pPHAB (FERM BP-3566), E.
coli K-12 [W3110 (tyrA)/pPHAD] (FERM BP-12659), E. coli K-12 [W3110
(tyrA)/pPHATerm] (FERM BP-12662) and E. coli K-12 [W3110
(tyrA)/pBR-aroG4, pACMAB] named as AJ 12604 (FERM BP-3579) may be
used (EP 488-424 B1). Furthermore, L-phenylalanine producing
bacteria belonging to the genus Escherichia which have an enhanced
activity of the protein encoded by the yedA gene or the yddG gene
may also be used (U.S. patent applications 2003/0148473 A1 and
2003/0157667 A1).
L-Tryptophan-Producing Bacteria
Examples of parent strains for deriving the L-tryptophan-producing
bacteria of the present invention include, but are not limited to,
strains belonging to the genus Escherichia, such as E. coli
JP4735/pMU3028 (DSM10122) and JP6015/pMU91 (DSM10123) deficient in
the tryptophanyl-tRNA synthetase encoded by mutant trpS gene (U.S.
Pat. No. 5,756,345); E. coli SV164 (pGH5) having a serA allele
encoding phosphoglycerate dehydrogenase resistant to feedback
inhibition by serine and a trpE allele encoding anthranilate
synthase resistant to feedback inhibition by tryptophan (U.S. Pat.
No. 6,180,373); E. coli AGX17 (pGX44) (NRRL B-12263) and
AGX6(pGX50) aroP (NRRL B-12264) deficient in the enzyme
tryptophanase (U.S. Pat. No. 4,371,614); E. coli AGX17/pGX50,
pACKG4-pps in which a phosphoenolpyruvate-producing ability is
enhanced (WO9708333, U.S. Pat. No. 6,319,696), and the like may be
used. Furthermore, L-tryptophan producing bacteria belonging to the
genus Escherichia which have an enhanced activity of the protein
encoded by the yedA gene or the yddG gene may also be used (U.S.
patent applications 2003/0148473 A1 and 2003/0157667 A1).
Examples of parent strains for deriving the L-tryptophan-producing
bacteria of the present invention also include strains in which one
or more activities of the enzymes selected from anthranilate
synthase (trpE), phosphoglycerate dehydrogenase (serA), and
tryptophan synthase (trpAB) are enhanced. The anthranilate synthase
and phosphoglycerate dehydrogenase are both subject to feedback
inhibition by L-tryptophan and L-serine, so a mutation which
results in desensitizing the feedback inhibition may be introduced
into these enzymes. Specific examples of strains having such a
mutation include an E. coli SV164 which harbors desensitized
anthranilate synthase and a strain obtained by transforming the
plasmid pGH5 into E. coli SV164 (WO 94/08031), which contains a
serA gene which has been mutated so that it encodes
feedback-desensitized phosphoglycerate dehydrogenase.
Examples of parent strains for deriving the L-tryptophan-producing
bacteria of the present invention also include strains transformed
with the tryptophan operon which contains a gene encoding
desensitized anthranilate synthase (JP 57-71397 A, JP 62-244382 A,
U.S. Pat. No. 4,371,614). Moreover, L-tryptophan-producing ability
may be imparted by enhancing expression of a gene which encodes
tryptophan synthase, among tryptophan operons (trpBA). The
tryptophan synthase consists of Ca and 13 subunits which are
encoded by trpA and trpB, respectively. In addition,
L-tryptophan-producing ability may be improved by enhancing
expression of the isocitrate lyase-malate synthase operon
(WO2005/103275).
L-Proline-Producing Bacteria
Examples of parent strains for deriving L-proline-producing
bacteria of the present invention include, but are not limited to,
strains belonging to the genus Escherichia, such as E. coli 702ilvA
(VKPM B-8012) which is deficient in the ilvA gene and is able to
produce L-proline (EP 1172433).
The bacterium of the present invention may be improved by enhancing
the expression of one or more genes involved in L-proline
biosynthesis. Examples of preferred genes for L-proline producing
bacteria include the proB gene coding for glutamate kinase which is
desensitized to feedback inhibition by L-proline (DE Patent
3127361). In addition, the bacterium of the present invention may
be improved by enhancing the expression of one or more genes coding
for proteins excreting L-amino acid from the bacterial cell. Such
genes include the b2682 and b2683 genes (ygaZH genes) (EP1239041
A2).
Examples of bacteria belonging to the genus Escherichia, which have
an activity to produce L-proline, include the following E. coli
strains: NRRL B-12403 and NRRL B-12404 (GB Patent 2075056), VKPM
B-8012 (Russian patent application 2000124295), plasmid mutants
described in DE Patent 3127361, plasmid mutants described by Bloom
F. R. et al (The 15th Miami winter symposium, 1983, p. 34), and the
like.
L-Arginine-Producing Bacteria
Examples of parent strains for deriving L-arginine-producing
bacteria of the present invention include, but are not limited to,
strains belonging to the genus Escherichia, such as E. coli strain
237 (VKPM B-7925) (U.S. Patent Application 2002/058315 A1) and its
derivative strains harboring mutant N-acetylglutamate synthase
(Russian Patent Application No. 2001112869), E. coli strain 382
(VKPM B-7926) (EP1170358A1), an arginine-producing strain into
which the argA gene encoding N-acetylglutamate synthetase is
introduced (EP1170361A1), and the like.
Examples of parent strains for deriving L-arginine producing
bacteria of the present invention also include strains in which
expression of one or more genes encoding an L-arginine biosynthetic
enzyme are enhanced. Examples of the L-arginine biosynthetic
enzymes include N-acetylglutamyl phosphate reductase (argC),
ornithine acetyl transferase (argJ), N-acetylglutamate kinase
(argB), acetylornithine transaminase (argD), ornithine carbamoyl
transferase (argF), argininosuccinic acid synthetase (argG),
argininosuccinic acid lyase (argH), and carbamoyl phosphate
synthetase (carAB).
L-Valine-Producing Bacteria
Example of parent strains for deriving L-valine-producing bacteria
of the present invention include, but are not limited to, strains
which have been modified to overexpress the ilvGMEDA operon (U.S.
Pat. No. 5,998,178). It is desirable to remove the region of the
ilvGMEDA operon which is required for attenuation so that
expression of the operon is not attenuated by the L-valine that is
produced. Furthermore, the ilvA gene in the operon is desirably
disrupted so that threonine deaminase activity is decreased.
Examples of parent strains for deriving L-valine-producing bacteria
of the present invention also include mutants of amino-acyl t-RNA
synthetase (U.S. Pat. No. 5,658,766). For example, E. coli VL1970,
which has a mutation in the ileS gene encoding isoleucine tRNA
synthetase, can be used. E. coli VL1970 has been deposited in the
Russian National Collection of Industrial Microorganisms (VKPM)
(Russia, 113545 Moscow, 1 Dorozhny Proezd.) on Jun. 24, 1988 under
accession number VKPM B-4411.
Furthermore, mutants requiring lipoic acid for growth and/or
lacking H.sup.+-ATPase can also be used (WO96/06926).
L-Isoleucine-Producing Bacteria
Examples of parent strains for deriving L-isoleucine producing
bacteria of the present invention include, but are not limited to,
mutants having resistance to 6-dimethylaminopurine (JP 5-304969 A),
mutants having resistance to an isoleucine analogue such as
thiaisoleucine and isoleucine hydroxamate, and mutants additionally
having resistance to DL-ethionine and/or arginine hydroxamate (JP
5-130882 A). In addition, recombinant strains transformed with
genes encoding proteins involved in L-isoleucine biosynthesis, such
as threonine deaminase and acetohydroxate synthase, can also be
used (JP 2-458 A, FR 0356739, and U.S. Pat. No. 5,998,178).
<1-2> Enhancement of the Iron Transporter Activity
The bacterium of the present invention can be obtained by modifying
a bacterium having an L-amino acid-producing ability as described
above so that the iron transporter activity is enhanced. However,
the L-amino acid-producing ability may be imparted after the
bacterium is modified so that the iron transporter activity is
enhanced. As described below, the iron transporter activity can be
enhanced by increasing the expression of a gene encoding a protein
involved in the tonB system, which can be achieved by enhancing the
expression of an endogenous gene by modifying an expression
regulatory region such as a promoter, or enhancing expression of an
exogenous gene by introducing a plasmid containing the gene, or the
like. In addition, these methods may be combined.
In the present invention, the term "iron transporter" means a
membrane protein which facilitates uptake of iron into the cellular
cytoplasm, and the phrase "modifying so that the iron transporter
activity is enhanced" includes when the number of iron transporter
molecules per cell increases and when the iron transporter activity
per molecule is improved as compared to a wild-type strain or
unmodified strain. The iron transporter activity is improved not
less than 150% per cell, preferably not less than 200%, more
preferably not less than 300% per cell as compared to a wild-type
strain or an unmodified strain. Examples of a wild-type strain
belonging to the Enterobacteriaceae family which can be used as a
control include Escherichia coli MG1655 strain (ATCC No. 47076),
W3110 strain (ATCC No. 27325), and Pantoea ananatis AJ13335 strain
(FERM BP-6615). The activity of the iron transporter can be
determined by measuring the uptake of labeled Fe.sup.3+ into the
cells (J. Bacteriol, May. 2003 p 1870-1885).
The iron transporter activity can be enhanced by enhancing the
expression of a gene encoding a protein involved in the tonB
system. The enhanced expression as compared to a wild-type or
unmodified strain can be confirmed by comparing the mRNA level of
the gene of the tonB system to that of a wild-type or unmodified
strain. Methods for confirming the expression of a gene include
Northern hybridization and RT-PCR (Molecular cloning (Cold spring
Harbor Laboratory Press, Cold spring Harbor (USA), 2001)). The
expression may be any level as long as it is increased as compared
to a wild-type or unmodified strain, and for example, the
expression is preferably increased not less than 1.5-fold, more
preferably not less than 2-fold, and further more preferably not
less than 3-fold as compared to a wild-type or unmodified strain.
Meanwhile, enhancing the expression of the gene of the tonB system
may also be confirmed by an increase in the level of the
corresponding protein as compared to a wild-type or unmodified
strain, and the protein level may be detected, for example, by
Western blotting using an antibody (Molecular cloning (Cold spring
Harbor Laboratory Press, Cold spring Harbor (USA), 2001)).
Examples of the gene encoding a protein involved in the tonB system
include the tonB gene, the fepA gene, and the fecA gene, or
homologues thereof. The tonB system is a system for uptake of iron
which is mediated by the membrane protein (TonB), and TonB also
regulates the activities of the iron transporters (FepA and FecA)
by transferring electrons to FepA and FecA (J. Bacteriol October
2001 vol. 183, No. 20, p 5885-5895). In the present invention,
examples of a gene of Escherichia coli include the tonB gene of SEQ
ID NO: 1 (nucleotide numbers 1309113 . . . 1309832 of GenBank
Accession No. NC-000913), the fepA gene of SEQ ID NO: 3 (a
complementary strand of nucleotide numbers 609477 . . . 611717 of
GenBank Accession No. NC.sub.--000913), and the fecA gene of SEQ ID
NO: 9 (a complementary strand of nucleotide numbers
4512376.451-4700 of GenBank Accession No. NC.sub.--000913.2).
In addition, the homologues of the above-mentioned E. coli genes
can be obtained by cloning, based on homologies to the above-listed
genes, from .gamma.-proteobacterium that belongs to the genus
Escherichia, Enterobacter, Klebsiella, Serratia, Erwinia, Yersinia,
or the like; a coryneform bacterium such as Corynebacterium
glutamicum, or Brevibacterium lactofermentum, a Pseudomonas
bacterium such as Pseudomonas aeruginosa; a Mycobacterium bacterium
such as Mycobacterium tuberculosis; or the like. The homologues may
be amplified by PCR using, for example, synthetic oligonucleotides
shown in SEQ ID NOS: 5 and 6 for the tonB gene, or SEQ ID NOS: 7
and 8 for the fepA gene.
The homologies between the amino acid sequences and nucleotide
sequences can be determined by using the algorithm BLAST developed
by Karlin and Altschul (Pro. Natl. Acad. Sci. USA, 90, 5873 (1993))
or the algorithm FASTA developed by Pearson (Methods Enzymol., 183,
63 (1990)). Based on the algorithm BLAST, programs called BLASTN
and BLASTX have been developed (http://www.ncbi.nlm.nih.gov).
The phrase "homologue of the gene of the tonB system" indicates
that a gene derived from other bacteria, or a naturally or
artificially mutated gene, which has high structural similarity to
the tonB gene, fepA gene, or fecA gene from Escherichia coli and is
able to improve the iron transport activity when introduced or
amplified in a host. The "homologues of the tonB gene, fepA gene,
and fecA gene which are involved in the tonB system" include genes
which encode a protein which has homology of at least 80%,
preferably at least 90%, more preferably 95%, particularly
preferably at least 98% to the entire sequence of SEQ ID NOS: 2
(tonB), 4 (fepA), or 10 (fecA), and is able to function as an iron
transporter regulatory factor (tonB) or as an iron transporter
(fepA or fecA). This function can be confirmed by expressing the
gene in a host cell and examining the transport of iron through the
cell membrane (see, the above-mentioned Non-patent documents 1 to
4). Alternatively, whether a gene is a homologue of the tonB gene,
fepA gene, or fecA gene can be confirmed by preparing a strain in
which the corresponding wild-type gene is disrupted and examining
whether the gene can complement the function of the wild-type gene
when introduced into the gene-disrupted strain, i.e., whether the
introduced gene can restore iron uptake.
Meanwhile, the tonB gene, fepA gene, and/or fecA gene are not
limited to their respective wild-type genes and may be mutants or
artificially modified genes that encode proteins having the amino
acid sequences of SEQ ID NO: 2, 4, or 10, but which may include
substitution, deletion, insertion, or addition of one or several
amino acids at one or a plurality of positions as long as the
function of the TonB, FepA, and/or FecA proteins encoded by these
genes is maintained, that is, the function as an iron transporter
regulatory factor (TonB) or an iron transporter (FepA or FecA). In
the present invention, although depending on the positions in the
ternary structure and types of amino acid residues in the proteins,
the term "one or several" specifically means 1 to 20, preferably 1
to 10, and more preferably 1 to 5. The above-mentioned substitution
is preferably a conservative substitution, and examples of
conservative substitutions include substitution between aromatic
amino acids such as a substitution among Phe, Trp, and Tyr;
substitution between hydrophobic amino acids such as a substitution
among Leu, Ile, and Val; substitution between polar amino acids
such as a substitution between Gln and Asn; substitution between
basic amino acids such as a substitution among Lys, Arg, and His;
substitution between acidic amino acids such as a substitution
between Asp and Glu; substitution between amino acids having a
hydroxyl group such as a substitution between Ser and Thr. Specific
examples of a conservative substitution include substitution of Ser
or Thr for Ala; substitution of Gln, His, or Lys for Arg;
substitution of Glu, Gln, Lys, His, or Asp for Asn; substitution of
Asn, Glu, or Gln for Asp; substitution of Ser or Ala for Cys;
substitution of Asn, Glu, Lys, His, Asp, or Arg for Gln;
substitution of Gly, Asn, Gln, Lys, or Asp for Glu; substitution of
Pro for Gly; substitution of Asn, Lys, Gln, Arg, or Tyr for His;
substitution of Leu, Met, Val, or Phe for Ile; substitution of Ile,
Met, Val, or Phe for Leu; substitution of Asn, Glu, Gln, His, or
Arg for Lys; substitution of Ile, Leu, Val, or Phe for Met;
substitution of Trp, Tyr, Met, Ile, or Leu for Phe; substitution of
Thr or Ala for Ser; substitution of Ser or Ala for Thr;
substitution of Phe or Tyr for Trp; substitution of His, Phe, or
Trp for Tyr; and substitution of Met, Ile, or Leu for Val.
Meanwhile, the above-mentioned amino acid substitution, deletion,
insertion, addition, or inversion may be a naturally occurring
mutation (mutant or variant) due to an individual difference, a
difference of types, or the like among the bacteria harboring the
tonB gene, fepA gene, or fecA gene.
Meanwhile, the tonB gene, fepA gene, and fecA gene may each be a
DNA which hybridizes with a nucleotide sequence complementary to
SEQ ID NOS: 1, 3, and 9, respectively, or a probe that can be
prepared from the sequence under stringent conditions, as long as
the gene encodes a protein having a function as an iron transporter
regulatory factor (tonB) or as an iron transporter (fepA or fecA).
In the present invention, the term "stringent conditions" refers to
conditions where a so-called specific hybrid is formed and
non-specific hybrid is not formed. It is difficult to clearly
define the conditions by a numerical value, and examples include
conditions where DNAs having high homology, for example, DNAs
having homology of at least 80%, preferably at least 90%, more
preferably at least 95%, or particularly preferably at least 98%
hybridize with each other and DNAs having homology of less than 80%
do not hybridize with each other; and specific examples thereof
include washing in general Southern hybridization, i.e., washing at
the salt concentration of 1.times.SSC, 0.1% SDS, preferably
0.1.times.SSC, 0.1% SDS, at 60.degree. C., preferably at 68.degree.
C., once, preferably twice or three times.
Expression of the above-mentioned tonB gene, fepA gene, and fecA
gene can be increased by, for example, increasing the copy number
of the genes in a cell using a gene recombination technique. For
example, a DNA fragment containing the gene is ligated to a vector
that functions in the host bacterium, preferably a multi-copy
vector, to thereby prepare a recombinant DNA, and the recombinant
DNA is used to transform the host bacterium.
When using the tonB gene and fepA gene of Escherichia coli, the
tonB gene and fepA gene can be obtained by PCR (polymerase chain
reaction; White, T. J. et al., Trends Genet. 5, 185 (1989)) using
primers based on the nucleotide sequences of SEQ ID NOS: 1, or 3,
for example, primers of SEQ ID NOS: 5 and 6 (tonB), or 7 and 8
(fepA) and a chromosomal DNA of Escherichia coli as the template.
The tonB gene and fepA gene from another bacterium can also be
obtained by PCR from the chromosomal DNA or genomic DNA library of
the bacterium using, as primers, oligonucleotides prepared based on
the known sequences of the tonB gene and fepA gene of the bacterium
or of the tonB gene and fepA gene of another kind of bacterium, or
the amino acid sequence of the TonB protein, and FepA protein; or
by hybridization using an oligonucleotide prepared based on the
sequence as a probe. A chromosomal DNA can be prepared from a
bacterium that serves as a DNA donor by the method of Saito and
Miura (Biochem. Biophys. Acta, 72, 619 (1963), Experiment Manual
for Biotechnology, edited by The Society for Biotechnology, Japan,
p 97-98, Baifukan Co., Ltd., 1992) or the like.
The fecA gene can be obtained in the same way.
Then, a recombinant DNA is prepared by ligating the tonB gene, fepA
gene, or fecA gene which has been amplified by PCR to a vector DNA
which is capable of functioning in the host bacterium. Examples of
the vector capable of functioning in the host bacterium include
vectors autonomously replicable in the host bacterium.
Examples of a vector which is autonomously replicable in
Escherichia coli include pUC19, pUC18, pHSG299, pHSG399, pHSG398,
pACYC184, (pHSG and pACYC are available from Takara Bio Inc.),
RSF1010 (Gene vol. 75(2), p 271-288, 1989), pBR322, pMW219, pMW119
(pMW is available form Nippon Gene Co., Ltd.), pSTV28, and pSTV29
(Takara Bio Inc.). A phage DNA vector can also be used.
To ligate the gene to the above-mentioned vector, the vector is
digested with a restriction enzyme corresponding to a recognition
site in the terminus of a DNA fragment containing the tonB gene,
fepA gene, and fecA gene. Ligation is generally performed using a
ligase such as T4 DNA ligase. Methods of digesting and ligating
DNA, preparation of a chromosomal DNA, preparation of a plasmid
DNA, transformation, PCR, design of oligonucleotides to be used as
primers are well known to the person skilled in the art. These
methods are described in Sambrook, J., Fritsch, E. F., and
Maniatis, T., "Molecular Cloning A Laboratory Manual, Second
Edition", Cold Sprig Harbor Laboratory Press, (1989), and the
like.
The thus-prepared recombinant DNA is introduced into a bacterium by
a conventional transformation method, such as electroporation
(Canadian Journal of Microbiology, 43, 197 (1997)). It is also
possible to increase the DNA permeability by treating the recipient
cells with calcium chloride, which has been reported for
Escherichia coli K-12 (Mandel, M. and Higa, A., J. Mol. Biol., 53,
159 (1970), and introduce a DNA into a competent cell at the
proliferation stage, which has been reported with Bacillus subtilis
(Duncan, C. H., Wilson, G. A and Young, F. E, Gene, 1, 153
(1977)).
The copy number of the tonB gene, fepA gene, and fecA gene can also
be increased by introducing multiple copies of the genes into the
chromosomal DNA of the host bacterium. Introducing multiple copies
of the genes into the chromosomal DNA of the host bacterium can be
attained by homologous recombination using a target sequence
present on the chromosomal DNA in multiple copies. This may be a
repetitive DNA or an inverted repeat present on the edge of a
transposing element. Alternatively, as disclosed in JP 2-109985 A,
multiple copies of the tonB gene, fepA gene, and fecA gene can be
introduced into the chromosomal DNA by inserting the gene into a
transposon, and transferring it so that multiple copies of the gene
are integrated into the chromosomal DNA. Integration of these genes
into the chromosome can be confirmed by Southern hybridization
using a portion of the genes as a probe.
Furthermore, expression of the tonB gene, fepA gene, and fecA gene
may be enhanced by, as described in WO 00/18935, substituting an
expression regulatory sequence such as the native promoter with a
stronger promoter, whether the gene is present on the chromosome or
a plasmid, amplifying a regulatory element that is able to increase
expression of the genes, or deleting or attenuating a regulatory
element that decreases expression of the genes. Examples of known
strong promoters include the lac promoter, trp promoter, trc
promoter, tac promoter, lambda phage PR promoter, PL promoter, and
tet promoter (WO98/004715).
Furthermore, the native promoter of the tonB gene, fepA gene, and
fecA gene can be strengthened by introducing nucleotide
substitution into the promoter (EP1033407). A method to evaluate
the strength of a promoter and examples of strong promoters are
described in Goldstein et al. (Prokaryotic promoters in
biotechnology. Biotechnol. Annu. Rev., 1995, 1, 105-128) or the
like. In addition, it is known that a spacer sequence between the
ribosome binding site (RBS) and the translation initiation codon,
especially, several nucleotides just upstream of the initiation
codon, has a great influence on translation efficiency. Therefore,
this sequence may be modified.
In addition, to enhance the activity of a protein encoded by the
tonB gene, fepA gene, and fecA gene, a mutation that increases the
activity of the iron transporter regulatory factor or iron
transporter may be introduced into the genes. Examples of such a
mutation include a mutation in the promoter sequence to increase
the transcription level of tonB gene, fepA gene, and fecA gene, and
a mutation in the coding region to increase the specific activities
of the TonB, FepA, or FecA proteins. In addition, a mutation to
enhance an activity of a protein that positively regulates the
expression of these genes may be introduced into the gene encoding
such a protein.
<2> Method of Producing L-Amino Acid
The method of producing an L-amino acid of the present invention is
to culture the bacterium of the present invention in a medium to
produce and accumulate an L-amino acid in the medium or bacterial
cells, and collecting the L-amino acid from the medium or the
bacterial cells.
Conventional media which are typically used in bacterial
fermentative production of an L-amino acid can be used. That is, a
general medium containing a carbon source, nitrogen source,
inorganic ion, and if necessary, other organic components can be
used. In the present invention, examples of the carbon source
include sugars such as glucose, sucrose, lactose, galactose,
fructose and a starch hydrolysate; alcohols such as glycerol and
sorbitol; and organic acids such as fumaric acid, citric acid and
succinic acid. Examples of the nitrogen source include inorganic
ammonium salts such as ammonium sulfate, ammonium chloride and
ammonium phosphate; an organic nitrogen such as a soybean
hydrolysate; ammonia gas; and aqueous ammonia. As organic trace
nutrients, auxotrophic substances such as vitamin B1 and
L-homoserine, yeast extract, and the like are preferably contained
in the medium in appropriate amounts. Besides such substances, if
necessary, potassium phosphate, magnesium sulfate, iron ion,
manganese ion, or the like may be added in small amounts. The
medium to be used in the present invention may be a natural medium
or a synthetic medium as long as it contains a carbon source,
nitrogen source, inorganic ion, and if necessary, other organic
trace nutrients.
The culture is preferably performed under aerobic conditions for 1
to 7 days at a temperature of 24.degree. C. to 37.degree. C. and a
pH of 5 to 9. The pH can be adjusted with an inorganic or organic
acidic or alkaline substance, ammonia gas or the like. The L-amino
acid can be collected from the fermentation liquid by a
conventional method such as ion-exchange resin, precipitation, and
other known methods. When the L-amino acid accumulates in the
bacterial cells, the L-amino acid can be collected, for example, by
disrupting the bacterial cells by ultrasonication or the like to
release the L-amino acid into the supernatant fraction, and then
the bacterial cells are removed by centrifugation, followed by
subjecting the resulting supernatant fraction to an ion-exchange
resin or the like.
When producing a basic L-amino acid, fermentation may be performed
while controlling the pH of the medium during culture to 6.5-9.0
and controlling the pH of the medium after completion of the
culture to 7.2-9.0, as well as controlling the pressure in the
fermentation tank during fermentation so that it is positive.
Alternatively, carbon dioxide or a mixed gas containing carbon
dioxide may be added to the medium so that a bicarbonate ion and/or
carbonate ion are present in an amount of at least 2 g/L in the
culture medium during the culture period. These ions function as
counter ions against the cation of the basic L-amino acids, and the
target basic L-amino acid can be collected (JP 2002-065287 A,
WO2006/038695).
EXAMPLES
Hereinafter, the present invention will be described in more detail
by referring to the following non-limiting examples. If not
otherwise specified, all the reagents used were purchased from Wako
Pure Chemical Industries, Ltd. or Nacalai Tesque, Inc. The
compositions of the media to be used in the Examples are shown
below. The pH of each medium was adjusted with NaOH or HCl.
(L Medium)
TABLE-US-00001 Bacto-tryptone (manufactured by Difco) 10 g/L Yeast
extract (manufactured by Difco) 5 g/L Sodium chloride 10 g/L pH
7.0
The medium was sterilized by steam at 120.degree. C. for 20
minutes.
[L Agar Medium]
TABLE-US-00002 L medium Bacto-agar 15 g/L
The medium was sterilized by steam at 120.degree. C. for 20
minutes.
[L-lysine production medium for Escherichia bacteria]
TABLE-US-00003 Glucose 40 g/L Ammonium sulfate 24 g/L Potassium
dihydrogen phosphate 1.0 g/L Magnesium sulfate heptahydrate 1.0 g/L
Iron sulfate heptahydrate 0.01 g/L Manganese sulfate heptahydrate
0.01 g/L Yeast extract 2.0 g/L Calcium carbonate (Official grade)
50 g/L (separately sterilized)
The medium was adjusted to pH 7.0 with potassium hydroxide and
sterilized by steam at 115.degree. C. for 10 minutes.
Glucose and magnesium sulfate heptahydrate were separately
sterilized.
Calcium carbonate (Official grade) was separately sterilized by
heating at 180.degree. C.
Chloramphenicol (25 mg/L) and ampicillin (100 mg/L) were added
before culture as antibiotics.
[L-Threonine Production Medium for Escherichia Bacteria]
TABLE-US-00004 Glucose 40 g/L Ammonium sulfate 16 g/L Potassium
dihydrogen phosphate 1.0 g/L Magnesium sulfate heptahydrate 1.0 g/L
Iron sulfate (IV) heptahydrate 0.01 g/L Manganese sulfate (IV)
heptahydrate 0.01 g/L Calcium carbonate (Official grade) 30 g/L
(separately sterilized)
The medium was adjusted to pH 7.5 with potassium hydroxide and
sterilized at 115.degree. C. for 10 minutes.
Glucose and magnesium sulfate heptahydrate were separately
sterilized.
Calcium carbonate (Official grade) was separately sterilized by
heating at 180.degree. C.
Streptomycin (100 mg/L) and ampicillin (100 mg/L) were added before
culture as antibiotics.
Example 1
<1> Construction of a Plasmid for Amplifying the tonB Gene or
the fepA Gene
To evaluate an effect of independent amplification of the tonB
gene, and fepA gene on production of L-lysine, plasmid vectors for
amplifying each of the genes were constructed. The entire
chromosomal nucleotide sequence of Escherichia coli (Escherichia
coli K-12 strain) has been disclosed (Science, 277, 1453-1474
(1997)), and primers to amplify the tonB gene and fepA gene were
designed based on the nucleotide sequences of the tonB gene
(nucleotide numbers 1309113-1309832 of GenBank Accession No.
NC.sub.--000913: SEQ ID NO: 1), and fepA gene (complementary strand
of nucleotide numbers 609477-611717 of NCBI Accession No.
NC.sub.--000913: SEQ ID NO: 3). SEQ ID NOS: 5 to 8 (for tonB: SEQ
ID NOS: 5 and 6; for fepA: SEQ ID NOS: 7 and 8) represent primers
to amplify the genes. These primers were used to perform PCR using
the chromosomal DNA of Escherichia coli MG1655 strain as a
template. The chromosomal DNA was obtained using Bacterial Genomic
DNA purification kit (Edge Bio Systems). PCR was performed using
pyrobest DNA polymerase (manufactured by Takara Bio Inc.) such that
a cycle of 96.degree. C. for 20 seconds, 65.degree. C. for 20
seconds, and 72.degree. C. for 2 minutes was repeated 25
cycles.
The amplified tonB gene and fepA gene were purified and ligated to
SmaI-digested vectors, pSTV28 (manufactured by Takara Bio Inc.) and
pMW119 (manufactured by Nippon Gene Co., Ltd.), respectively, to
thereby obtain a plasmid for amplifying the tonB gene (pStonB), and
a plasmid for amplifying the fepA gene (pSfepA).
Example 2
Construction of a Strain in which the Lysine Decarboxylase-Encoding
Genes (cadA and ldcC) are Disrupted
A strain which produces no lysine decarboxylase was constructed.
The lysine decarboxylases are encoded by the cadA gene (Genbank
Accession No. NP.sub.--418555, SEQ ID NO: 17) and the ldcC gene
(Genbank Accession No. NP.sub.--414728, SEQ ID NO: 11) (WO
96/17930). WC196 (FERM BP-5252) was the parent strain.
The cadA gene and the ldcC gene were disrupted by the method
developed by Datsenko and Wanner, which is called "Red-driven
integration" (Proc. Natl. Acad. Sci. USA, 2000, vol. 97, No. 12, p
6640-6645) and by an excision system derived from .lamda. phage (J.
Bacteriol. 2002 September; 184(18): 5200-3. Interactions between
integrase and excisionase in the phage lambda excisive
nucleoprotein complex. Cho E H, Gumport R I, Gardner J F.).
"Red-driven integration" makes it possible to construct a
gene-disrupted strain in one step by employing a PCR product
obtained by using as primers synthetic oligonucleotides designed to
have a part of the targeted gene on the 5'-ends and a part of an
antibiotic-resistance gene on the 3'-ends. Combining the .lamda.
phage-derived excision system permits the removal of the
antibiotic-resistance gene that has been incorporated into the
gene-disrupted strain (WO2005/010175).
(1) Disruption of the cadA Gene
The pMW118-attL-Cm-attR plasmid (WO2005/010175) was used as a
template for PCR. pMW118-attL-Cm-attR was obtained by inserting the
attL and attR genes, which are attachment sites for .lamda. phage,
and the cat gene, which is an antibiotic resistance gene, into
pMW118 (Takara Bio Inc.) The genes are arranged in the following
order: attL-cat-attR.
PCR was performed using, as primers, the synthetic oligonucleotides
shown in SEQ ID NOS: 13 and 14, which have sequences corresponding
to attL and attR on the 3'-ends and a sequence corresponding to a
part of the targeted cadA gene on the 5'-ends.
The amplified PCR product was purified on an agarose gel and
introduced into the Escherichia coli WC1-96 strain by
electroporation. This strain harbors pKD46 which has
temperature-sensitive replicability. pKD46 (Proc. Natl. Acad. Sci.
USA, 2000, vol. 97, No. 12, p 6640-6645) contains a DNA fragment of
2,154 nucleotides derived from .lamda. phage which contains the Red
recombinase-encoding genes (.gamma., .beta., and exo genes) of the
.lamda. Red homologous recombination system, which is controlled by
an arabinose-inducible ParaB promoter (GenBank/EMBL Accession No.
J02459, nucleotide numbers 31088 to 33241). pKD46 is necessary to
integrate the PCR product into the chromosome of the WC1-96
strain.
Competent cells for electroporation were prepared as follows. That
is, cells of the Escherichia coli WC1-96 strain were cultured
overnight at 30.degree. C. in LB medium containing 100 mg/L
ampicillin, and then diluted 100-fold with 5 mL of SOB medium
(Molecular Cloning: Laboratory manual, 2nd edition, Sambrook, J. et
al., Cold Spring Harbor Laboratory Press (1989)) containing
ampicillin (20 mg/L) and L-arabinose (1 mM). The diluted cells were
grown with aeration at 30.degree. C. until the OD600 reached about
0.6, and then concentrated 100-fold and washed three times with 10%
glycerol so that the cells were available for electroporation. The
electroporation was performed with 70 .mu.L of the competent cells
and about 100 ng of the PCR product. After the electroporation, 1
mL of SOC medium (Molecular Cloning: Laboratory manual, 2nd
edition, Sambrook, J. et al., Cold Spring Harbor Laboratory Press
(1989)) was added to the cells, and cells were cultured at
37.degree. C. for 2.5 hours, and then subjected to plate culture
onto L-agar medium containing Cm (chloramphenicol) (25 mg/L), to
thereby select Cm-resistant recombinant strains. Subsequently, to
remove the plasmid pKD46, the cells were subcultured twice at
42.degree. C. on L-agar medium containing Cm, and ampicillin
resistance of the resultant colonies were examined, to thereby
yield ampicillin-sensitive strains in which the pKD46 was
cured.
Deletion of the cadA gene in the mutant strain, which had been
identified by the chloramphenicol-resistance gene, was confirmed by
PCR. The cadA-disrupted strain was named
WC196.DELTA.cadA:att-cat.
Subsequently, the helper plasmid pMW-intxis-ts (WO2005/010175) was
used to remove the att-cat gene which had been introduced into the
cadA gene. The plasmid pMW-intxis-ts carries a gene encoding the
integrase (Int) of .lamda. phage, and the gene encoding excisionase
(Xis), and has temperature-sensitive replicability.
Competent cells of the WC196.DELTA.cadA:att-cat strain were
prepared by a conventional method, and were then transformed with
the helper plasmid pMW-intxis-ts, and then subjected to plate
culture at 30.degree. C. onto L-agar medium containing 50 mg/L
ampicillin, to thereby select ampicillin-resistant strains.
Subsequently, to remove the plasmid pMW-intxis-ts, the cells were
subcultured twice at 42.degree. C. on L-agar medium, and ampicillin
resistance and chloramphenicol resistance of the resulting colonies
were examined, to thereby yield a chloramphenicol and
ampicillin-sensitive strain, in which the cadA gene was disrupted,
and att-cat and the pMW-intxis-ts were removed. The strain was
named WC196.DELTA.cadA.
(2) Disruption of the ldcC Gene in the WC196.DELTA.cadA Strain
The ldcC gene in the WC196.DELTA.cadA strain was disrupted by using
oligonucleotides of SEQ ID NOS: 15 and 16 as primers in the same
way as described above. In this way, a cadA and ldcC-disrupted
strain named WC196.DELTA.cadA.DELTA.ldcC was obtained.
<2> Introduction of a Plasmid for Lysine Production into the
WC196.DELTA.cadA.DELTA.ldcC Strain
WC196.DELTA.cadA.DELTA.ldcC strain was transformed with a plasmid
for lysine production named pCABD2 (WO 01/53459), which carries the
dapA gene, dapB gene, lysC gene and ddh gene, to thereby yield the
WC196.DELTA.cadA.DELTA.ldcC/pCABD2 strain (WC196LC/pCABD2).
<2-2> Effect of Amplification of the tonB Gene in an
L-Lysine-Producing Strain of Escherichia Bacterium
The WC196LC/pCABD2 strain was transformed with the plasmid for
amplifying the tonB gene (pStonB) which was constructed in Example
1 and a control plasmid (pSTV28) (Takara Bio Inc), and
chloramphenicol-resistant strains were selected. Introduction of
the plasmids was confirmed, and the pStonB-introduced strain and
pSTV28-introduced strain were named WC196LC/pCABD2/pStonB strain
and WC196LC/pCABD2/pSTV28 strain, respectively.
WC196LC/pCABD2/pStonB strain and WC196LC/pCABD2/pSTV28 strain were
cultured at 37.degree. C. in L-medium containing 50 mg/L
chloramphenicol until the final OD600 reached about 0.6, and then
an equal volume of 40% glycerol solution was added to the culture,
followed by stirring. Then, the resulting suspension was dispensed
in appropriate amounts and stored at -80.degree. C., which was used
as a glycerol stock.
The glycerol stocks of the strains were thawed, and 100 .mu.L of
each strain was uniformly applied on an L-plate containing 25 mg/L
chloramphenicol and 20 mg/L streptomycin, and cultured at
37.degree. C. for 24 hours. The bacterial cells growing on the
plate were suspended in 2-3 mL of a fermentation medium so that
OD620 became 13.5, and 1 mL of the suspension was inoculated into
20 mL of the fermentation medium (L-lysine production medium for
Escherichia bacteria) containing 25 mg/L chloramphenicol and 20
mg/L streptomycin in a 500 mL-Sakaguchi flask and cultured at
37.degree. C. using a reciprocal shaker for 48 hours. The amount of
L-lysine which accumulated in the medium was determined using a
Biotech Analyzer AS210 (Sakura Seiki Co. Ltd.).
Table 1 shows the amounts of L-lysine present after 24 hours. In
the case of the WC196LC/pCABD2/pStonB strain, the amount of
L-lysine present 24 hours later was higher as compared to the
WC196LC/pCABD2/pSTV28 strain, which did not contain the tonB gene.
This data shows that the L-lysine-producing ability was improved by
enhancing the expression of the tonB gene.
TABLE-US-00005 TABLE 1 L-lysine (g/L) Bacterial strain present
after 24 hours WC196LC/pCABD2/pSTV28 7.0 WC196LC/pCABD2/pStonB
9.5
<2-3> Effect of Amplification of the fepA Gene in L-Lysine
Producing Strain of Escherichia Bacterium
WC196LC/pCABD2 strain was transformed with the plasmid for
amplifying fepA gene (pSfepA) which was constructed in Example 1,
and an ampicillin-resistant strain was selected. Introduction of
the plasmid pSfepA was confirmed, and the pSfepA-introduced strain
was named WC196LC/pCABD2/pSfepA.
In the same way as <2-2>, glycerol stocks of
WC196LC/pCABD2/pSfepA strain and WC196LC/pCABD2 strain as a control
were prepared. Here, 100 mg/L ampicillin was used instead of 50
mg/L chloramphenicol.
The glycerol stocks of these strains were thawed, and 100 .mu.L of
each of the strains was uniformly applied on an L-plate containing
100 mg/L ampicillin and 20 mg/L streptomycin, followed by culture
at 37.degree. C. for 24 hours. The bacterial cells which grew on
the plate were collected and inoculated into the fermentation
medium in the same way as <2-2> except that 100 mg/L
ampicillin was used instead of 50 mg/L chloramphenicol, and the
culture was performed over 24 hours, followed by determination of
the L-lysine amount using a Biotech Analyzer AS210 (Sakura Seiki
Co., Ltd.).
Table 2 shows the amount of L-lysine which was present after 24
hours. For the WC196LC/pCABD2/pSfepA strain, the amount of L-lysine
present after 24 hours was higher as compared to the WC196LC/pCABD2
strain, which did not contain the fepA gene, which revealed that
the L-lysine-producing ability was improved by enhancing the
expression of the fepA gene.
TABLE-US-00006 TABLE 2 L-lysine (g/L) Bacterial strain present
after 24 hours WC196LC/pCABD2 7.3 WC196LC/pCABD2/pSfepA 9.6
Example 3
Effect of Amplification of the fepA Gene in L-Threonine-Producing
Strain of Escherichia Bacterium
B-5318 strain was used as an L-threonine-producing strain. B-5318
strain has been deposited in Russian National Collection of
Industrial Microorganisms (VKPM), GNII Genetika) on May 3, 1990,
under accession No. VKPM B-5318. Construction of a fepA
gene-amplified strain from the L-threonine producing bacterium was
performed using the plasmid pSfepA which was constructed in Example
1.
The B-5318 strain was transformed with the plasmid pSfepA, and an
ampicillin-resistant strain was selected. Introduction of the
plasmid was confirmed, and the pSfepA-introduced strain was named
B5318/pSfepA.
The B5318/pSfepA strain, and the B5318/pMW strain into which a
control plasmid was introduced were cultured at 37.degree. C. in an
L-medium containing 100 mg/L ampicillin and 100 mg/L streptomycin
until the final OD600 reached about 0.6, and then an equal volume
of 40% glycerol solution was added to the obtained culture,
followed by stirring. Then, the mixture was dispensed in
appropriate amounts and stored in glycerol at -80.degree. C., which
was used as a glycerol stock.
The glycerol stocks of the strains were thawed, and 100 .mu.L of
each of the strains was uniformly applied on an L-plate containing
100 mg/L ampicillin and 100 mg/L streptomycin, and cultured at
37.degree. C. for 24 hours. The bacterial cells growing on the
plate were suspended in 6 mL of physiological saline so that OD620
became 3.0, and 0.5 mL of each of the suspensions was inoculated
into 20 mL of the fermentation medium (L-threonine-producing medium
for Escherichia bacteria) containing 100 mg/L ampicillin and 100
mg/L streptomycin in a 500 mL-Sakaguchi flask, and cultured at
37.degree. C. for 24 hours using a reciprocal shaker. After the
culture, the amount of L-threonine which had accumulated in the
medium was determined using high performance liquid
chromatography.
Table 3 shows the amount of L-threonine present after 24 hours. For
the B5318/pSfepA strain, the amount of L-threonine was higher as
compared to the B5318/pMW strain, which revealed that the
productivity of L-threonine was improved by enhancing the
expression of the fepA gene.
TABLE-US-00007 TABLE 3 L-threonine (g/L) Bacterial strain present
after 24 hours B5318/pMW 4.22 B5318/pSfepA 4.87
INDUSTRIAL APPLICABILITY
Use of the bacterium of the present invention enables efficient
fermentative production of basic L-amino acids such as L-lysine,
L-ornithine, L-arginine, L-histidine and L-citrulline; aliphatic
L-amino acids such as L-isoleucine, L-alanine, L-valine, L-leucine
and L-glycine; hydroxy monoaminocarboxylic acids such as
L-threonine and L-serine; cyclic L-amino acid such as L-proline;
aromatic L-amino acids such as L-phenylalanine, L-tyrosine and
L-tryptophan; sulfur-containing L-amino acids such as L-cysteine,
L-cystine and L-methionine; and acidic L-amino acids such as
L-glutamic acid, L-aspartic acid, L-glutamine and L-asparagine.
While the invention has been described in detail with reference to
preferred embodiments thereof, it will be apparent to one skilled
in the art that various changes can be made, and equivalents
employed, without departing from the scope of the invention. Each
of the aforementioned documents is incorporated by reference herein
in its entirety.
SEQUENCE LISTINGS
1
181720DNAEscherichia coliCDS(1)..(717) 1atg acc ctt gat tta cct cgc
cgc ttc ccc tgg ccg acg tta ctt tcg 48Met Thr Leu Asp Leu Pro Arg
Arg Phe Pro Trp Pro Thr Leu Leu Ser1 5 10 15gtc tgc att cat ggt gct
gtt gtg gcg ggt ctg ctc tat acc tcg gta 96Val Cys Ile His Gly Ala
Val Val Ala Gly Leu Leu Tyr Thr Ser Val 20 25 30cat cag gtt att gaa
cta cct gcg cct gcg cag ccg att tct gtc acg 144His Gln Val Ile Glu
Leu Pro Ala Pro Ala Gln Pro Ile Ser Val Thr 35 40 45atg gtt acg cct
gct gat ctc gaa ccg cca caa gcc gtt cag ccg cca 192Met Val Thr Pro
Ala Asp Leu Glu Pro Pro Gln Ala Val Gln Pro Pro 50 55 60ccg gag ccg
gtg gta gag cca gaa ccg gaa cct gag ccg atc ccc gaa 240Pro Glu Pro
Val Val Glu Pro Glu Pro Glu Pro Glu Pro Ile Pro Glu65 70 75 80ccg
cca aaa gaa gca ccg gtg gtc att gaa aag ccg aag ccg aaa cct 288Pro
Pro Lys Glu Ala Pro Val Val Ile Glu Lys Pro Lys Pro Lys Pro 85 90
95aag cca aaa ccg aag ccg gtg aaa aag gta cag gag cag cca aaa cgc
336Lys Pro Lys Pro Lys Pro Val Lys Lys Val Gln Glu Gln Pro Lys Arg
100 105 110gat gtc aaa ccc gta gag tcg cgt ccg gca tca ccg ttt gaa
aat acg 384Asp Val Lys Pro Val Glu Ser Arg Pro Ala Ser Pro Phe Glu
Asn Thr 115 120 125gca ccg gca cgc ctg aca tca agt aca gca acg gct
gca acc agc aag 432Ala Pro Ala Arg Leu Thr Ser Ser Thr Ala Thr Ala
Ala Thr Ser Lys 130 135 140ccg gtt acc agt gtg gct tca gga cca cgc
gca tta agc cgt aat cag 480Pro Val Thr Ser Val Ala Ser Gly Pro Arg
Ala Leu Ser Arg Asn Gln145 150 155 160ccg cag tat ccg gca cga gca
cag gca ttg cgc att gaa ggg cag gtt 528Pro Gln Tyr Pro Ala Arg Ala
Gln Ala Leu Arg Ile Glu Gly Gln Val 165 170 175aaa gtt aaa ttt gac
gtt acg ccg gat ggt cgc gtg gat aac gta caa 576Lys Val Lys Phe Asp
Val Thr Pro Asp Gly Arg Val Asp Asn Val Gln 180 185 190atc ctc tca
gcc aag cct gcg aac atg ttt gag cgt gag gtg aaa aat 624Ile Leu Ser
Ala Lys Pro Ala Asn Met Phe Glu Arg Glu Val Lys Asn 195 200 205gcg
atg cgc aga tgg cgt tat gag ccg ggt aag cca ggc agt ggg att 672Ala
Met Arg Arg Trp Arg Tyr Glu Pro Gly Lys Pro Gly Ser Gly Ile 210 215
220gtg gtg aat atc ctg ttt aaa att aac ggc acc acc gaa att cag taa
720Val Val Asn Ile Leu Phe Lys Ile Asn Gly Thr Thr Glu Ile Gln225
230 2352239PRTEscherichia coli 2Met Thr Leu Asp Leu Pro Arg Arg Phe
Pro Trp Pro Thr Leu Leu Ser1 5 10 15Val Cys Ile His Gly Ala Val Val
Ala Gly Leu Leu Tyr Thr Ser Val 20 25 30His Gln Val Ile Glu Leu Pro
Ala Pro Ala Gln Pro Ile Ser Val Thr 35 40 45Met Val Thr Pro Ala Asp
Leu Glu Pro Pro Gln Ala Val Gln Pro Pro 50 55 60Pro Glu Pro Val Val
Glu Pro Glu Pro Glu Pro Glu Pro Ile Pro Glu65 70 75 80Pro Pro Lys
Glu Ala Pro Val Val Ile Glu Lys Pro Lys Pro Lys Pro 85 90 95Lys Pro
Lys Pro Lys Pro Val Lys Lys Val Gln Glu Gln Pro Lys Arg 100 105
110Asp Val Lys Pro Val Glu Ser Arg Pro Ala Ser Pro Phe Glu Asn Thr
115 120 125Ala Pro Ala Arg Leu Thr Ser Ser Thr Ala Thr Ala Ala Thr
Ser Lys 130 135 140Pro Val Thr Ser Val Ala Ser Gly Pro Arg Ala Leu
Ser Arg Asn Gln145 150 155 160Pro Gln Tyr Pro Ala Arg Ala Gln Ala
Leu Arg Ile Glu Gly Gln Val 165 170 175Lys Val Lys Phe Asp Val Thr
Pro Asp Gly Arg Val Asp Asn Val Gln 180 185 190Ile Leu Ser Ala Lys
Pro Ala Asn Met Phe Glu Arg Glu Val Lys Asn 195 200 205Ala Met Arg
Arg Trp Arg Tyr Glu Pro Gly Lys Pro Gly Ser Gly Ile 210 215 220Val
Val Asn Ile Leu Phe Lys Ile Asn Gly Thr Thr Glu Ile Gln225 230
23532241DNAEscherichia coliCDS(1)..(2238) 3atg aac aag aag att cat
tcc ctg gcc ttg ttg gtc aat ctg ggg att 48Met Asn Lys Lys Ile His
Ser Leu Ala Leu Leu Val Asn Leu Gly Ile1 5 10 15tat ggg gta gcg cag
gca caa gag ccg acc gat act cct gtt tca cat 96Tyr Gly Val Ala Gln
Ala Gln Glu Pro Thr Asp Thr Pro Val Ser His 20 25 30gac gat act att
gtc gtt acc gcc gcc gag cag aac tta cag gcg cct 144Asp Asp Thr Ile
Val Val Thr Ala Ala Glu Gln Asn Leu Gln Ala Pro 35 40 45ggc gtt tcg
acc atc acc gca gat gaa atc cgc aaa aac ccg gtt gcc 192Gly Val Ser
Thr Ile Thr Ala Asp Glu Ile Arg Lys Asn Pro Val Ala 50 55 60cgc gat
gtg tcg aag atc atc cgt acc atg cca ggc gtt aac ctg acc 240Arg Asp
Val Ser Lys Ile Ile Arg Thr Met Pro Gly Val Asn Leu Thr65 70 75
80ggt aac tcc acc agt ggt cag cgt ggg aat aac cga cag att gat att
288Gly Asn Ser Thr Ser Gly Gln Arg Gly Asn Asn Arg Gln Ile Asp Ile
85 90 95cgc ggt atg ggt ccg gaa aac acg ctg att ttg att gac ggc aag
ccg 336Arg Gly Met Gly Pro Glu Asn Thr Leu Ile Leu Ile Asp Gly Lys
Pro 100 105 110gta agc agc cgt aac tcg gtg cgt cag ggc tgg cgt ggc
gag cgc gat 384Val Ser Ser Arg Asn Ser Val Arg Gln Gly Trp Arg Gly
Glu Arg Asp 115 120 125acc cgt ggt gat act tcc tgg gtg cca cct gaa
atg att gaa cgt att 432Thr Arg Gly Asp Thr Ser Trp Val Pro Pro Glu
Met Ile Glu Arg Ile 130 135 140gaa gtt ctg cgt ggt ccg gca gct gcg
cgt tat ggc aac ggc gcg gcg 480Glu Val Leu Arg Gly Pro Ala Ala Ala
Arg Tyr Gly Asn Gly Ala Ala145 150 155 160ggc ggc gtg gtt aac atc
att acc aaa aaa ggc agc ggc gag tgg cac 528Gly Gly Val Val Asn Ile
Ile Thr Lys Lys Gly Ser Gly Glu Trp His 165 170 175ggc tcc tgg gac
gca tat ttc aat gcg cca gaa cat aaa gag gaa ggt 576Gly Ser Trp Asp
Ala Tyr Phe Asn Ala Pro Glu His Lys Glu Glu Gly 180 185 190gcc acc
aaa cgc act aac ttt agc ctg acc ggt ccg ctg ggc gac gaa 624Ala Thr
Lys Arg Thr Asn Phe Ser Leu Thr Gly Pro Leu Gly Asp Glu 195 200
205ttc agc ttc cgt ttg tat ggc aac ctc gac aaa acc cag gct gac gcg
672Phe Ser Phe Arg Leu Tyr Gly Asn Leu Asp Lys Thr Gln Ala Asp Ala
210 215 220tgg gat atc aac cag ggc cat cag tcc gcg cgt gcc gga acg
tat gcc 720Trp Asp Ile Asn Gln Gly His Gln Ser Ala Arg Ala Gly Thr
Tyr Ala225 230 235 240acg acg tta cca gcc ggg cgc gaa ggg gta atc
aac aaa gat att aat 768Thr Thr Leu Pro Ala Gly Arg Glu Gly Val Ile
Asn Lys Asp Ile Asn 245 250 255ggc gtg gtg cgc tgg gat ttc gcg cca
ttg caa tcg ctg gaa ctg gaa 816Gly Val Val Arg Trp Asp Phe Ala Pro
Leu Gln Ser Leu Glu Leu Glu 260 265 270gca ggt tac agc cgc cag ggt
aac ctg tat gcg ggc gac acc cag aat 864Ala Gly Tyr Ser Arg Gln Gly
Asn Leu Tyr Ala Gly Asp Thr Gln Asn 275 280 285acc aac tcc gat tcc
tat acc cgc tcg aaa tat ggc gat gaa acc aac 912Thr Asn Ser Asp Ser
Tyr Thr Arg Ser Lys Tyr Gly Asp Glu Thr Asn 290 295 300cgt ctg tat
cgc cag aac tac gcg ctg acc tgg aac ggt ggc tgg gat 960Arg Leu Tyr
Arg Gln Asn Tyr Ala Leu Thr Trp Asn Gly Gly Trp Asp305 310 315
320aac ggc gtg acc acc agc aac tgg gtg cag tac gaa cac acc cgt aac
1008Asn Gly Val Thr Thr Ser Asn Trp Val Gln Tyr Glu His Thr Arg Asn
325 330 335tcg cgt att ccg gaa ggt ctg gcg ggc ggt acc gaa ggg aaa
ttt aac 1056Ser Arg Ile Pro Glu Gly Leu Ala Gly Gly Thr Glu Gly Lys
Phe Asn 340 345 350gaa aaa gcg aca cag gat ttc gtc gat atc gat ctt
gat gac gtg atg 1104Glu Lys Ala Thr Gln Asp Phe Val Asp Ile Asp Leu
Asp Asp Val Met 355 360 365ctg cac agc gaa gtt aac ctg ccg att gat
ttc ctc gtt aac cag acg 1152Leu His Ser Glu Val Asn Leu Pro Ile Asp
Phe Leu Val Asn Gln Thr 370 375 380ctg acg ctg ggt acg gag tgg aat
cag caa cgg atg aag gac tta agt 1200Leu Thr Leu Gly Thr Glu Trp Asn
Gln Gln Arg Met Lys Asp Leu Ser385 390 395 400tcc aac acc cag gca
ctg acc gga acg aat acc ggt ggc gct att gat 1248Ser Asn Thr Gln Ala
Leu Thr Gly Thr Asn Thr Gly Gly Ala Ile Asp 405 410 415ggc gtg agt
acc acc gac cgt agc ccg tat tca aaa gca gaa att ttc 1296Gly Val Ser
Thr Thr Asp Arg Ser Pro Tyr Ser Lys Ala Glu Ile Phe 420 425 430tcg
ctg ttt gcc gaa aac aac atg gag ctg act gac agc acc atc gta 1344Ser
Leu Phe Ala Glu Asn Asn Met Glu Leu Thr Asp Ser Thr Ile Val 435 440
445acg ccg ggg ctg cgt ttc gat cat cac agt att gtc ggc aat aac tgg
1392Thr Pro Gly Leu Arg Phe Asp His His Ser Ile Val Gly Asn Asn Trp
450 455 460agc ccg gcg ctg aac ata tcg caa ggt tta ggc gat gac ttc
acg ctg 1440Ser Pro Ala Leu Asn Ile Ser Gln Gly Leu Gly Asp Asp Phe
Thr Leu465 470 475 480aaa atg ggc atc gcc cgt gct tat aaa gcg ccg
agc ctg tac cag act 1488Lys Met Gly Ile Ala Arg Ala Tyr Lys Ala Pro
Ser Leu Tyr Gln Thr 485 490 495aac ccg aac tac att ctc tac agt aaa
ggt cag ggt tgc tat gcc agc 1536Asn Pro Asn Tyr Ile Leu Tyr Ser Lys
Gly Gln Gly Cys Tyr Ala Ser 500 505 510gcg ggc ggc tgc tat ctg caa
ggt aac gat gac ctg aaa gca gaa acc 1584Ala Gly Gly Cys Tyr Leu Gln
Gly Asn Asp Asp Leu Lys Ala Glu Thr 515 520 525agc atc aac aaa gag
att ggt ctg gag ttc aaa cgc gac ggg tgg ctg 1632Ser Ile Asn Lys Glu
Ile Gly Leu Glu Phe Lys Arg Asp Gly Trp Leu 530 535 540gcg ggc gtc
acc tgg ttc cgt aac gat tat cgc aat aag att gaa gcg 1680Ala Gly Val
Thr Trp Phe Arg Asn Asp Tyr Arg Asn Lys Ile Glu Ala545 550 555
560ggc tat gtg gct gta ggg caa aac gca gtc ggc acc gat ctc tat cag
1728Gly Tyr Val Ala Val Gly Gln Asn Ala Val Gly Thr Asp Leu Tyr Gln
565 570 575tgg gat aac gtg ccg aaa gcg gtg gtt gaa ggt ctg gaa gga
tcg tta 1776Trp Asp Asn Val Pro Lys Ala Val Val Glu Gly Leu Glu Gly
Ser Leu 580 585 590aac gta ccg gtt agc gaa acg gtg atg tgg acc aat
aac atc act tat 1824Asn Val Pro Val Ser Glu Thr Val Met Trp Thr Asn
Asn Ile Thr Tyr 595 600 605atg ctg aag agt gaa aac aaa acc acg ggc
gac cgt ttg tcg atc atc 1872Met Leu Lys Ser Glu Asn Lys Thr Thr Gly
Asp Arg Leu Ser Ile Ile 610 615 620ccg gag tat acg ttg aac tca acg
ctg agc tgg cag gca cgg gaa gat 1920Pro Glu Tyr Thr Leu Asn Ser Thr
Leu Ser Trp Gln Ala Arg Glu Asp625 630 635 640ttg tcg atg caa acg
acc ttc acc tgg tac ggc aag cag cag ccg aag 1968Leu Ser Met Gln Thr
Thr Phe Thr Trp Tyr Gly Lys Gln Gln Pro Lys 645 650 655aag tac aac
tat aaa ggt cag cca gcg gtt gga ccg gaa acc aaa gaa 2016Lys Tyr Asn
Tyr Lys Gly Gln Pro Ala Val Gly Pro Glu Thr Lys Glu 660 665 670att
agt cct tac agc att gtt ggc ctg agc gcg acc tgg gat gtg acg 2064Ile
Ser Pro Tyr Ser Ile Val Gly Leu Ser Ala Thr Trp Asp Val Thr 675 680
685aag aat gtc agt ctg acc ggc ggc gtg gac aat ctg ttc gac aaa cgt
2112Lys Asn Val Ser Leu Thr Gly Gly Val Asp Asn Leu Phe Asp Lys Arg
690 695 700ttg tgg cgt gcg ggt aat gcc cag acc acg ggc gat ttg gca
ggg gcc 2160Leu Trp Arg Ala Gly Asn Ala Gln Thr Thr Gly Asp Leu Ala
Gly Ala705 710 715 720aac tat atc gcc ggt gcc ggg gcg tat acc tat
aac gag ccg gga cgt 2208Asn Tyr Ile Ala Gly Ala Gly Ala Tyr Thr Tyr
Asn Glu Pro Gly Arg 725 730 735acg tgg tat atg agc gta aac acc cac
ttc tga 2241Thr Trp Tyr Met Ser Val Asn Thr His Phe 740
7454746PRTEscherichia coli 4Met Asn Lys Lys Ile His Ser Leu Ala Leu
Leu Val Asn Leu Gly Ile1 5 10 15Tyr Gly Val Ala Gln Ala Gln Glu Pro
Thr Asp Thr Pro Val Ser His 20 25 30Asp Asp Thr Ile Val Val Thr Ala
Ala Glu Gln Asn Leu Gln Ala Pro 35 40 45Gly Val Ser Thr Ile Thr Ala
Asp Glu Ile Arg Lys Asn Pro Val Ala 50 55 60Arg Asp Val Ser Lys Ile
Ile Arg Thr Met Pro Gly Val Asn Leu Thr65 70 75 80Gly Asn Ser Thr
Ser Gly Gln Arg Gly Asn Asn Arg Gln Ile Asp Ile 85 90 95Arg Gly Met
Gly Pro Glu Asn Thr Leu Ile Leu Ile Asp Gly Lys Pro 100 105 110Val
Ser Ser Arg Asn Ser Val Arg Gln Gly Trp Arg Gly Glu Arg Asp 115 120
125Thr Arg Gly Asp Thr Ser Trp Val Pro Pro Glu Met Ile Glu Arg Ile
130 135 140Glu Val Leu Arg Gly Pro Ala Ala Ala Arg Tyr Gly Asn Gly
Ala Ala145 150 155 160Gly Gly Val Val Asn Ile Ile Thr Lys Lys Gly
Ser Gly Glu Trp His 165 170 175Gly Ser Trp Asp Ala Tyr Phe Asn Ala
Pro Glu His Lys Glu Glu Gly 180 185 190Ala Thr Lys Arg Thr Asn Phe
Ser Leu Thr Gly Pro Leu Gly Asp Glu 195 200 205Phe Ser Phe Arg Leu
Tyr Gly Asn Leu Asp Lys Thr Gln Ala Asp Ala 210 215 220Trp Asp Ile
Asn Gln Gly His Gln Ser Ala Arg Ala Gly Thr Tyr Ala225 230 235
240Thr Thr Leu Pro Ala Gly Arg Glu Gly Val Ile Asn Lys Asp Ile Asn
245 250 255Gly Val Val Arg Trp Asp Phe Ala Pro Leu Gln Ser Leu Glu
Leu Glu 260 265 270Ala Gly Tyr Ser Arg Gln Gly Asn Leu Tyr Ala Gly
Asp Thr Gln Asn 275 280 285Thr Asn Ser Asp Ser Tyr Thr Arg Ser Lys
Tyr Gly Asp Glu Thr Asn 290 295 300Arg Leu Tyr Arg Gln Asn Tyr Ala
Leu Thr Trp Asn Gly Gly Trp Asp305 310 315 320Asn Gly Val Thr Thr
Ser Asn Trp Val Gln Tyr Glu His Thr Arg Asn 325 330 335Ser Arg Ile
Pro Glu Gly Leu Ala Gly Gly Thr Glu Gly Lys Phe Asn 340 345 350Glu
Lys Ala Thr Gln Asp Phe Val Asp Ile Asp Leu Asp Asp Val Met 355 360
365Leu His Ser Glu Val Asn Leu Pro Ile Asp Phe Leu Val Asn Gln Thr
370 375 380Leu Thr Leu Gly Thr Glu Trp Asn Gln Gln Arg Met Lys Asp
Leu Ser385 390 395 400Ser Asn Thr Gln Ala Leu Thr Gly Thr Asn Thr
Gly Gly Ala Ile Asp 405 410 415Gly Val Ser Thr Thr Asp Arg Ser Pro
Tyr Ser Lys Ala Glu Ile Phe 420 425 430Ser Leu Phe Ala Glu Asn Asn
Met Glu Leu Thr Asp Ser Thr Ile Val 435 440 445Thr Pro Gly Leu Arg
Phe Asp His His Ser Ile Val Gly Asn Asn Trp 450 455 460Ser Pro Ala
Leu Asn Ile Ser Gln Gly Leu Gly Asp Asp Phe Thr Leu465 470 475
480Lys Met Gly Ile Ala Arg Ala Tyr Lys Ala Pro Ser Leu Tyr Gln Thr
485 490 495Asn Pro Asn Tyr Ile Leu Tyr Ser Lys Gly Gln Gly Cys Tyr
Ala Ser 500 505 510Ala Gly Gly Cys Tyr Leu Gln Gly Asn Asp Asp Leu
Lys Ala Glu Thr 515 520 525Ser Ile Asn Lys Glu Ile Gly Leu Glu Phe
Lys Arg Asp Gly Trp Leu 530 535 540Ala Gly Val Thr Trp Phe Arg Asn
Asp Tyr Arg Asn Lys Ile Glu Ala545 550 555 560Gly Tyr Val Ala Val
Gly Gln Asn Ala Val Gly Thr Asp Leu Tyr Gln 565 570 575Trp Asp Asn
Val Pro Lys Ala Val Val Glu Gly Leu Glu Gly Ser Leu 580 585 590Asn
Val Pro Val Ser Glu Thr Val Met Trp Thr Asn Asn Ile Thr Tyr 595 600
605Met Leu Lys Ser Glu Asn Lys Thr Thr Gly Asp Arg Leu Ser Ile Ile
610 615 620Pro Glu Tyr Thr Leu Asn Ser Thr Leu Ser Trp Gln Ala Arg
Glu Asp625 630
635 640Leu Ser Met Gln Thr Thr Phe Thr Trp Tyr Gly Lys Gln Gln Pro
Lys 645 650 655Lys Tyr Asn Tyr Lys Gly Gln Pro Ala Val Gly Pro Glu
Thr Lys Glu 660 665 670Ile Ser Pro Tyr Ser Ile Val Gly Leu Ser Ala
Thr Trp Asp Val Thr 675 680 685Lys Asn Val Ser Leu Thr Gly Gly Val
Asp Asn Leu Phe Asp Lys Arg 690 695 700Leu Trp Arg Ala Gly Asn Ala
Gln Thr Thr Gly Asp Leu Ala Gly Ala705 710 715 720Asn Tyr Ile Ala
Gly Ala Gly Ala Tyr Thr Tyr Asn Glu Pro Gly Arg 725 730 735Thr Trp
Tyr Met Ser Val Asn Thr His Phe 740 745521DNAArtificial
sequence5'-primer for tonB 5atttgaaagg gcgaagatct g
21621DNAArtificial sequence3'-primer for tonB 6ttgatcctga
aggaaaacct c 21720DNAArtificial sequence5'-primer for fepA
7ccaccagaaa gtgacctcaa 20821DNAArtificial sequence3'-primer for
fepA 8ccagagtaaa tcctgctcac a 2192325DNAEscherichia
coliCDS(1)..(2322) 9atg acg ccg tta cgc gtt ttt cgt aaa aca aca cct
ttg gtt aac acc 48Met Thr Pro Leu Arg Val Phe Arg Lys Thr Thr Pro
Leu Val Asn Thr1 5 10 15att cgc ctg agc ctg ctg ccg ctg gcc ggt ctc
tcg ttt tcc gct ttt 96Ile Arg Leu Ser Leu Leu Pro Leu Ala Gly Leu
Ser Phe Ser Ala Phe 20 25 30gct gca cag gtt aat atc gca ccg gga tcg
ctc gat aaa gcg ctc aat 144Ala Ala Gln Val Asn Ile Ala Pro Gly Ser
Leu Asp Lys Ala Leu Asn 35 40 45cag tat gcc gca cac agc gga ttt acc
ctc tcg gtt gac gcc agc ctg 192Gln Tyr Ala Ala His Ser Gly Phe Thr
Leu Ser Val Asp Ala Ser Leu 50 55 60acg cgc ggc aag cag agc aac ggc
ctg cac ggc gat tac gac gtc gag 240Thr Arg Gly Lys Gln Ser Asn Gly
Leu His Gly Asp Tyr Asp Val Glu65 70 75 80agc ggc ctg caa caa ctg
ctg gac ggc agc gga ctg cag gta aaa ccg 288Ser Gly Leu Gln Gln Leu
Leu Asp Gly Ser Gly Leu Gln Val Lys Pro 85 90 95ctg gga aat aac agc
tgg acg ctg gag ccc gcg ccc gca cca aaa gaa 336Leu Gly Asn Asn Ser
Trp Thr Leu Glu Pro Ala Pro Ala Pro Lys Glu 100 105 110gat gcc ctg
acc gtg gtc ggc gac tgg ctg ggt gat gcg cgt gaa aac 384Asp Ala Leu
Thr Val Val Gly Asp Trp Leu Gly Asp Ala Arg Glu Asn 115 120 125gac
gta ttt gaa cat gct ggc gcg cgt gac gtg atc cgc cgt gag gat 432Asp
Val Phe Glu His Ala Gly Ala Arg Asp Val Ile Arg Arg Glu Asp 130 135
140ttc gcc aaa acc ggc gca acc acc atg cgt gag gta ctt aac cgc atc
480Phe Ala Lys Thr Gly Ala Thr Thr Met Arg Glu Val Leu Asn Arg
Ile145 150 155 160cct ggc gtc agc gcg ccg gaa aac aac ggc acc ggc
agc cac gac ctg 528Pro Gly Val Ser Ala Pro Glu Asn Asn Gly Thr Gly
Ser His Asp Leu 165 170 175gcg atg aac ttt ggc atc cgg ggc ctg aac
ccg cgc ctc gcc agc cgc 576Ala Met Asn Phe Gly Ile Arg Gly Leu Asn
Pro Arg Leu Ala Ser Arg 180 185 190tcg acc gtc ctg atg gac ggc atc
ccc gtc ccc ttc gcc cct tac ggt 624Ser Thr Val Leu Met Asp Gly Ile
Pro Val Pro Phe Ala Pro Tyr Gly 195 200 205cag ccg cag ctt tca ctg
gct ccc gtt tcg ctc ggc aac atg gat gcc 672Gln Pro Gln Leu Ser Leu
Ala Pro Val Ser Leu Gly Asn Met Asp Ala 210 215 220att gac gtg gta
cgc ggt ggt ggt gcg gtg cgt tac gga ccg cag agc 720Ile Asp Val Val
Arg Gly Gly Gly Ala Val Arg Tyr Gly Pro Gln Ser225 230 235 240gtg
ggc ggc gtg gtg aac ttt gtt acc cgt gcc att ccg cag gac ttt 768Val
Gly Gly Val Val Asn Phe Val Thr Arg Ala Ile Pro Gln Asp Phe 245 250
255ggt atc gag gcg ggc gtg gaa ggt cag ctc agc cca acc tct tca caa
816Gly Ile Glu Ala Gly Val Glu Gly Gln Leu Ser Pro Thr Ser Ser Gln
260 265 270aac aac ccg aaa gag acg cac aac ctg atg gtg ggc ggc aca
gcg gac 864Asn Asn Pro Lys Glu Thr His Asn Leu Met Val Gly Gly Thr
Ala Asp 275 280 285aac ggt ttt ggc acc gcg ctg ctc tac tcc ggc acg
cgc ggc agt gac 912Asn Gly Phe Gly Thr Ala Leu Leu Tyr Ser Gly Thr
Arg Gly Ser Asp 290 295 300tgg cgc gag cac agc gcc acc cgc atc gac
gac ctg atg ctg aaa agc 960Trp Arg Glu His Ser Ala Thr Arg Ile Asp
Asp Leu Met Leu Lys Ser305 310 315 320aaa tat gcg ccg gat gag gtg
cac acc ttc aac agc ctg ctg caa tat 1008Lys Tyr Ala Pro Asp Glu Val
His Thr Phe Asn Ser Leu Leu Gln Tyr 325 330 335tac gac ggt gaa gcc
gac atg ccc ggt ggc ctg tct cgc gcg gat tac 1056Tyr Asp Gly Glu Ala
Asp Met Pro Gly Gly Leu Ser Arg Ala Asp Tyr 340 345 350gac gcc gat
cgc tgg caa tcc acc cgc ccg tat gac cgc ttc tgg ggt 1104Asp Ala Asp
Arg Trp Gln Ser Thr Arg Pro Tyr Asp Arg Phe Trp Gly 355 360 365cgt
cgc aag ctg gcg agc ctg ggc tac cag ttc cag cca gac agc cag 1152Arg
Arg Lys Leu Ala Ser Leu Gly Tyr Gln Phe Gln Pro Asp Ser Gln 370 375
380cat aaa ttc aac att cag ggg ttc tac acc caa acc ctg cgc agc ggc
1200His Lys Phe Asn Ile Gln Gly Phe Tyr Thr Gln Thr Leu Arg Ser
Gly385 390 395 400tac ctg gag caa ggc aaa cgc atc acc ctc tcg ccg
cgt aac tac tgg 1248Tyr Leu Glu Gln Gly Lys Arg Ile Thr Leu Ser Pro
Arg Asn Tyr Trp 405 410 415gtg cgc ggt att gag cca cgc tac agc cag
atc ttt atg atc ggc cct 1296Val Arg Gly Ile Glu Pro Arg Tyr Ser Gln
Ile Phe Met Ile Gly Pro 420 425 430tcc gcg cac gaa gtg ggc gtg ggc
tat cgc tat ttg aat gaa tca acg 1344Ser Ala His Glu Val Gly Val Gly
Tyr Arg Tyr Leu Asn Glu Ser Thr 435 440 445cat gaa atg cgt tac tac
acc gcc acc agc agc ggg cag ttg ccg tcc 1392His Glu Met Arg Tyr Tyr
Thr Ala Thr Ser Ser Gly Gln Leu Pro Ser 450 455 460ggc tca agc cct
tac gac cgc gat acg cgt tcc ggc acc gag gcg cac 1440Gly Ser Ser Pro
Tyr Asp Arg Asp Thr Arg Ser Gly Thr Glu Ala His465 470 475 480gcc
tgg tat ctg gat gac aaa atc gac atc ggc aac tgg acc atc acg 1488Ala
Trp Tyr Leu Asp Asp Lys Ile Asp Ile Gly Asn Trp Thr Ile Thr 485 490
495ccg ggt atg cgt ttc gaa cat atc gag tca tac cag aac aac gcc atc
1536Pro Gly Met Arg Phe Glu His Ile Glu Ser Tyr Gln Asn Asn Ala Ile
500 505 510aca ggc acg cac gaa gaa gtg agc tat aac gca ccg ctt ccg
gcg ttg 1584Thr Gly Thr His Glu Glu Val Ser Tyr Asn Ala Pro Leu Pro
Ala Leu 515 520 525aac gtg ctc tat cac ctg act gac agc tgg aat ctt
tat gca aac act 1632Asn Val Leu Tyr His Leu Thr Asp Ser Trp Asn Leu
Tyr Ala Asn Thr 530 535 540gaa ggc tcg ttc ggc acc gta cag tac agc
cag att ggc aag gct gtg 1680Glu Gly Ser Phe Gly Thr Val Gln Tyr Ser
Gln Ile Gly Lys Ala Val545 550 555 560caa agc ggc aat gtt gaa ccg
gaa aaa gcg cga acc tgg gaa ctc ggt 1728Gln Ser Gly Asn Val Glu Pro
Glu Lys Ala Arg Thr Trp Glu Leu Gly 565 570 575acc cgc tac gac gac
ggc gcg ctg acg gcg gaa atg ggg ctg ttc ctg 1776Thr Arg Tyr Asp Asp
Gly Ala Leu Thr Ala Glu Met Gly Leu Phe Leu 580 585 590att aac ttt
aac aat cag tac gac tcc aac cag acc aac gac acc gtc 1824Ile Asn Phe
Asn Asn Gln Tyr Asp Ser Asn Gln Thr Asn Asp Thr Val 595 600 605act
gca cgt ggc aaa acg cgc cat acc ggg ctg gaa acg cag gca cgt 1872Thr
Ala Arg Gly Lys Thr Arg His Thr Gly Leu Glu Thr Gln Ala Arg 610 615
620tac gat ctg ggt acg cta acg cca acg ctt gat aac gtt tcc atc tac
1920Tyr Asp Leu Gly Thr Leu Thr Pro Thr Leu Asp Asn Val Ser Ile
Tyr625 630 635 640gcc agc tat gcg tat gtg aac gcg gaa atc cgc gag
aaa ggc gac acc 1968Ala Ser Tyr Ala Tyr Val Asn Ala Glu Ile Arg Glu
Lys Gly Asp Thr 645 650 655tac ggc aat ctg gta cca ttc tcc ccg aaa
cat aaa ggc acg ctg ggc 2016Tyr Gly Asn Leu Val Pro Phe Ser Pro Lys
His Lys Gly Thr Leu Gly 660 665 670gtg gac tac aag cca gga aac tgg
acg ttc aat ctg aac agc gat ttc 2064Val Asp Tyr Lys Pro Gly Asn Trp
Thr Phe Asn Leu Asn Ser Asp Phe 675 680 685cag tcc agc cag ttt gcg
gat aac gcc aat acg gtg aaa gag agc gcc 2112Gln Ser Ser Gln Phe Ala
Asp Asn Ala Asn Thr Val Lys Glu Ser Ala 690 695 700gac ggc agt acc
ggc cgc att ccc ggc ttc atg ctc tgg ggc gca cgc 2160Asp Gly Ser Thr
Gly Arg Ile Pro Gly Phe Met Leu Trp Gly Ala Arg705 710 715 720gtg
gcg tat gac ttt ggc ccg cag atg gca gat ctg aac ctg gca ttc 2208Val
Ala Tyr Asp Phe Gly Pro Gln Met Ala Asp Leu Asn Leu Ala Phe 725 730
735ggt gtg aaa aac atc ttc gac cag gac tac ttc atc cgc tct tat gac
2256Gly Val Lys Asn Ile Phe Asp Gln Asp Tyr Phe Ile Arg Ser Tyr Asp
740 745 750gac aac aac aaa ggc atc tat gca ggc cag ccg cgc acg ctg
tat atg 2304Asp Asn Asn Lys Gly Ile Tyr Ala Gly Gln Pro Arg Thr Leu
Tyr Met 755 760 765cag ggg tcg ttg aag ttc tga 2325Gln Gly Ser Leu
Lys Phe 77010774PRTEscherichia coli 10Met Thr Pro Leu Arg Val Phe
Arg Lys Thr Thr Pro Leu Val Asn Thr1 5 10 15Ile Arg Leu Ser Leu Leu
Pro Leu Ala Gly Leu Ser Phe Ser Ala Phe 20 25 30Ala Ala Gln Val Asn
Ile Ala Pro Gly Ser Leu Asp Lys Ala Leu Asn 35 40 45Gln Tyr Ala Ala
His Ser Gly Phe Thr Leu Ser Val Asp Ala Ser Leu 50 55 60Thr Arg Gly
Lys Gln Ser Asn Gly Leu His Gly Asp Tyr Asp Val Glu65 70 75 80Ser
Gly Leu Gln Gln Leu Leu Asp Gly Ser Gly Leu Gln Val Lys Pro 85 90
95Leu Gly Asn Asn Ser Trp Thr Leu Glu Pro Ala Pro Ala Pro Lys Glu
100 105 110Asp Ala Leu Thr Val Val Gly Asp Trp Leu Gly Asp Ala Arg
Glu Asn 115 120 125Asp Val Phe Glu His Ala Gly Ala Arg Asp Val Ile
Arg Arg Glu Asp 130 135 140Phe Ala Lys Thr Gly Ala Thr Thr Met Arg
Glu Val Leu Asn Arg Ile145 150 155 160Pro Gly Val Ser Ala Pro Glu
Asn Asn Gly Thr Gly Ser His Asp Leu 165 170 175Ala Met Asn Phe Gly
Ile Arg Gly Leu Asn Pro Arg Leu Ala Ser Arg 180 185 190Ser Thr Val
Leu Met Asp Gly Ile Pro Val Pro Phe Ala Pro Tyr Gly 195 200 205Gln
Pro Gln Leu Ser Leu Ala Pro Val Ser Leu Gly Asn Met Asp Ala 210 215
220Ile Asp Val Val Arg Gly Gly Gly Ala Val Arg Tyr Gly Pro Gln
Ser225 230 235 240Val Gly Gly Val Val Asn Phe Val Thr Arg Ala Ile
Pro Gln Asp Phe 245 250 255Gly Ile Glu Ala Gly Val Glu Gly Gln Leu
Ser Pro Thr Ser Ser Gln 260 265 270Asn Asn Pro Lys Glu Thr His Asn
Leu Met Val Gly Gly Thr Ala Asp 275 280 285Asn Gly Phe Gly Thr Ala
Leu Leu Tyr Ser Gly Thr Arg Gly Ser Asp 290 295 300Trp Arg Glu His
Ser Ala Thr Arg Ile Asp Asp Leu Met Leu Lys Ser305 310 315 320Lys
Tyr Ala Pro Asp Glu Val His Thr Phe Asn Ser Leu Leu Gln Tyr 325 330
335Tyr Asp Gly Glu Ala Asp Met Pro Gly Gly Leu Ser Arg Ala Asp Tyr
340 345 350Asp Ala Asp Arg Trp Gln Ser Thr Arg Pro Tyr Asp Arg Phe
Trp Gly 355 360 365Arg Arg Lys Leu Ala Ser Leu Gly Tyr Gln Phe Gln
Pro Asp Ser Gln 370 375 380His Lys Phe Asn Ile Gln Gly Phe Tyr Thr
Gln Thr Leu Arg Ser Gly385 390 395 400Tyr Leu Glu Gln Gly Lys Arg
Ile Thr Leu Ser Pro Arg Asn Tyr Trp 405 410 415Val Arg Gly Ile Glu
Pro Arg Tyr Ser Gln Ile Phe Met Ile Gly Pro 420 425 430Ser Ala His
Glu Val Gly Val Gly Tyr Arg Tyr Leu Asn Glu Ser Thr 435 440 445His
Glu Met Arg Tyr Tyr Thr Ala Thr Ser Ser Gly Gln Leu Pro Ser 450 455
460Gly Ser Ser Pro Tyr Asp Arg Asp Thr Arg Ser Gly Thr Glu Ala
His465 470 475 480Ala Trp Tyr Leu Asp Asp Lys Ile Asp Ile Gly Asn
Trp Thr Ile Thr 485 490 495Pro Gly Met Arg Phe Glu His Ile Glu Ser
Tyr Gln Asn Asn Ala Ile 500 505 510Thr Gly Thr His Glu Glu Val Ser
Tyr Asn Ala Pro Leu Pro Ala Leu 515 520 525Asn Val Leu Tyr His Leu
Thr Asp Ser Trp Asn Leu Tyr Ala Asn Thr 530 535 540Glu Gly Ser Phe
Gly Thr Val Gln Tyr Ser Gln Ile Gly Lys Ala Val545 550 555 560Gln
Ser Gly Asn Val Glu Pro Glu Lys Ala Arg Thr Trp Glu Leu Gly 565 570
575Thr Arg Tyr Asp Asp Gly Ala Leu Thr Ala Glu Met Gly Leu Phe Leu
580 585 590Ile Asn Phe Asn Asn Gln Tyr Asp Ser Asn Gln Thr Asn Asp
Thr Val 595 600 605Thr Ala Arg Gly Lys Thr Arg His Thr Gly Leu Glu
Thr Gln Ala Arg 610 615 620Tyr Asp Leu Gly Thr Leu Thr Pro Thr Leu
Asp Asn Val Ser Ile Tyr625 630 635 640Ala Ser Tyr Ala Tyr Val Asn
Ala Glu Ile Arg Glu Lys Gly Asp Thr 645 650 655Tyr Gly Asn Leu Val
Pro Phe Ser Pro Lys His Lys Gly Thr Leu Gly 660 665 670Val Asp Tyr
Lys Pro Gly Asn Trp Thr Phe Asn Leu Asn Ser Asp Phe 675 680 685Gln
Ser Ser Gln Phe Ala Asp Asn Ala Asn Thr Val Lys Glu Ser Ala 690 695
700Asp Gly Ser Thr Gly Arg Ile Pro Gly Phe Met Leu Trp Gly Ala
Arg705 710 715 720Val Ala Tyr Asp Phe Gly Pro Gln Met Ala Asp Leu
Asn Leu Ala Phe 725 730 735Gly Val Lys Asn Ile Phe Asp Gln Asp Tyr
Phe Ile Arg Ser Tyr Asp 740 745 750Asp Asn Asn Lys Gly Ile Tyr Ala
Gly Gln Pro Arg Thr Leu Tyr Met 755 760 765Gln Gly Ser Leu Lys Phe
770112142DNAEscherichia coliCDS(1)..(2139) 11atg aac atc att gcc
att atg gga ccg cat ggc gtc ttt tat aaa gat 48Met Asn Ile Ile Ala
Ile Met Gly Pro His Gly Val Phe Tyr Lys Asp1 5 10 15gag ccc atc aaa
gaa ctg gag tcg gcg ctg gtg gcg caa ggc ttt cag 96Glu Pro Ile Lys
Glu Leu Glu Ser Ala Leu Val Ala Gln Gly Phe Gln 20 25 30att atc tgg
cca caa aac agc gtt gat ttg ctg aaa ttt atc gag cat 144Ile Ile Trp
Pro Gln Asn Ser Val Asp Leu Leu Lys Phe Ile Glu His 35 40 45aac cct
cga att tgc ggc gtg att ttt gac tgg gat gag tac agt ctc 192Asn Pro
Arg Ile Cys Gly Val Ile Phe Asp Trp Asp Glu Tyr Ser Leu 50 55 60gat
tta tgt agc gat atc aat cag ctt aat gaa tat ctc ccg ctt tat 240Asp
Leu Cys Ser Asp Ile Asn Gln Leu Asn Glu Tyr Leu Pro Leu Tyr65 70 75
80gcc ttc atc aac acc cac tcg acg atg gat gtc agc gtg cag gat atg
288Ala Phe Ile Asn Thr His Ser Thr Met Asp Val Ser Val Gln Asp Met
85 90 95cgg atg gcg ctc tgg ttt ttt gaa tat gcg ctg ggg cag gcg gaa
gat 336Arg Met Ala Leu Trp Phe Phe Glu Tyr Ala Leu Gly Gln Ala Glu
Asp 100 105 110atc gcc att cgt atg cgt cag tac acc gac gaa tat ctt
gat aac att 384Ile Ala Ile Arg Met Arg Gln Tyr Thr Asp Glu Tyr Leu
Asp Asn Ile 115 120 125aca ccg ccg ttc acg aaa gcc ttg ttt acc tac
gtc aaa gag cgg aag 432Thr Pro Pro Phe Thr Lys Ala Leu Phe Thr Tyr
Val Lys Glu Arg Lys 130 135 140tac acc ttt tgt acg ccg ggg cat atg
ggc ggc acc gca tat caa aaa 480Tyr Thr Phe Cys Thr Pro Gly His Met
Gly Gly Thr Ala Tyr Gln Lys145 150 155 160agc ccg gtt ggc tgt ctg
ttt tat gat ttt ttc
ggc ggg aat act ctt 528Ser Pro Val Gly Cys Leu Phe Tyr Asp Phe Phe
Gly Gly Asn Thr Leu 165 170 175aag gct gat gtc tct att tcg gtc acc
gag ctt ggt tcg ttg ctc gac 576Lys Ala Asp Val Ser Ile Ser Val Thr
Glu Leu Gly Ser Leu Leu Asp 180 185 190cac acc ggg cca cac ctg gaa
gcg gaa gag tac atc gcg cgg act ttt 624His Thr Gly Pro His Leu Glu
Ala Glu Glu Tyr Ile Ala Arg Thr Phe 195 200 205ggc gcg gaa cag agt
tat atc gtt acc aac gga aca tcg acg tcg aac 672Gly Ala Glu Gln Ser
Tyr Ile Val Thr Asn Gly Thr Ser Thr Ser Asn 210 215 220aaa att gtg
ggt atg tac gcc gcg cca tcc ggc agt acg ctg ttg atc 720Lys Ile Val
Gly Met Tyr Ala Ala Pro Ser Gly Ser Thr Leu Leu Ile225 230 235
240gac cgc aat tgt cat aaa tcg ctg gcg cat ctg ttg atg atg aac gat
768Asp Arg Asn Cys His Lys Ser Leu Ala His Leu Leu Met Met Asn Asp
245 250 255gta gtg cca gtc tgg ctg aaa ccg acg cgt aat gcg ttg ggg
att ctt 816Val Val Pro Val Trp Leu Lys Pro Thr Arg Asn Ala Leu Gly
Ile Leu 260 265 270ggt ggg atc ccg cgc cgt gaa ttt act cgc gac agc
atc gaa gag aaa 864Gly Gly Ile Pro Arg Arg Glu Phe Thr Arg Asp Ser
Ile Glu Glu Lys 275 280 285gtc gct gct acc acg caa gca caa tgg ccg
gtt cat gcg gtg atc acc 912Val Ala Ala Thr Thr Gln Ala Gln Trp Pro
Val His Ala Val Ile Thr 290 295 300aac tcc acc tat gat ggc ttg ctc
tac aac acc gac tgg atc aaa cag 960Asn Ser Thr Tyr Asp Gly Leu Leu
Tyr Asn Thr Asp Trp Ile Lys Gln305 310 315 320acg ctg gat gtc ccg
tcg att cac ttc gat tct gcc tgg gtg ccg tac 1008Thr Leu Asp Val Pro
Ser Ile His Phe Asp Ser Ala Trp Val Pro Tyr 325 330 335acc cat ttt
cat ccg atc tac cag ggt aaa agt ggt atg agc ggc gag 1056Thr His Phe
His Pro Ile Tyr Gln Gly Lys Ser Gly Met Ser Gly Glu 340 345 350cgt
gtt gcg gga aaa gtg atc ttc gaa acg caa tcg acc cac aaa atg 1104Arg
Val Ala Gly Lys Val Ile Phe Glu Thr Gln Ser Thr His Lys Met 355 360
365ctg gcg gcg tta tcg cag gct tcg ctg atc cac att aaa ggc gag tat
1152Leu Ala Ala Leu Ser Gln Ala Ser Leu Ile His Ile Lys Gly Glu Tyr
370 375 380gac gaa gag gcc ttt aac gaa gcc ttt atg atg cat acc acc
acc tcg 1200Asp Glu Glu Ala Phe Asn Glu Ala Phe Met Met His Thr Thr
Thr Ser385 390 395 400ccc agt tat ccc att gtt gct tcg gtt gag acg
gcg gcg gcg atg ctg 1248Pro Ser Tyr Pro Ile Val Ala Ser Val Glu Thr
Ala Ala Ala Met Leu 405 410 415cgt ggt aat ccg ggc aaa cgg ctg att
aac cgt tca gta gaa cga gct 1296Arg Gly Asn Pro Gly Lys Arg Leu Ile
Asn Arg Ser Val Glu Arg Ala 420 425 430ctg cat ttt cgc aaa gag gtc
cag cgg ctg cgg gaa gag tct gac ggt 1344Leu His Phe Arg Lys Glu Val
Gln Arg Leu Arg Glu Glu Ser Asp Gly 435 440 445tgg ttt ttc gat atc
tgg caa ccg ccg cag gtg gat gaa gcc gaa tgc 1392Trp Phe Phe Asp Ile
Trp Gln Pro Pro Gln Val Asp Glu Ala Glu Cys 450 455 460tgg ccc gtt
gcg cct ggc gaa cag tgg cac ggc ttt aac gat gcg gat 1440Trp Pro Val
Ala Pro Gly Glu Gln Trp His Gly Phe Asn Asp Ala Asp465 470 475
480gcc gat cat atg ttt ctc gat ccg gtt aaa gtc act att ttg aca ccg
1488Ala Asp His Met Phe Leu Asp Pro Val Lys Val Thr Ile Leu Thr Pro
485 490 495ggg atg gac gag cag ggc aat atg agc gag gag ggg atc ccg
gcg gcg 1536Gly Met Asp Glu Gln Gly Asn Met Ser Glu Glu Gly Ile Pro
Ala Ala 500 505 510ctg gta gca aaa ttc ctc gac gaa cgt ggg atc gta
gta gag aaa acc 1584Leu Val Ala Lys Phe Leu Asp Glu Arg Gly Ile Val
Val Glu Lys Thr 515 520 525ggc cct tat aac ctg ctg ttt ctc ttt agt
att ggc atc gat aaa acc 1632Gly Pro Tyr Asn Leu Leu Phe Leu Phe Ser
Ile Gly Ile Asp Lys Thr 530 535 540aaa gca atg gga tta ttg cgt ggg
ttg acg gaa ttc aaa cgc tct tac 1680Lys Ala Met Gly Leu Leu Arg Gly
Leu Thr Glu Phe Lys Arg Ser Tyr545 550 555 560gat ctc aac ctg cgg
atc aaa aat atg cta ccc gat ctc tat gca gaa 1728Asp Leu Asn Leu Arg
Ile Lys Asn Met Leu Pro Asp Leu Tyr Ala Glu 565 570 575gat ccc gat
ttc tac cgc aat atg cgt att cag gat ctg gca caa ggg 1776Asp Pro Asp
Phe Tyr Arg Asn Met Arg Ile Gln Asp Leu Ala Gln Gly 580 585 590atc
cat aag ctg att cgt aaa cac gat ctt ccc ggt ttg atg ttg cgg 1824Ile
His Lys Leu Ile Arg Lys His Asp Leu Pro Gly Leu Met Leu Arg 595 600
605gca ttc gat act ttg ccg gag atg atc atg acg cca cat cag gca tgg
1872Ala Phe Asp Thr Leu Pro Glu Met Ile Met Thr Pro His Gln Ala Trp
610 615 620caa cga caa att aaa ggc gaa gta gaa acc att gcg ctg gaa
caa ctg 1920Gln Arg Gln Ile Lys Gly Glu Val Glu Thr Ile Ala Leu Glu
Gln Leu625 630 635 640gtc ggt aga gta tcg gca aat atg atc ctg cct
tat cca ccg ggc gta 1968Val Gly Arg Val Ser Ala Asn Met Ile Leu Pro
Tyr Pro Pro Gly Val 645 650 655ccg ctg ttg atg cct gga gaa atg ctg
acc aaa gag agc cgc aca gta 2016Pro Leu Leu Met Pro Gly Glu Met Leu
Thr Lys Glu Ser Arg Thr Val 660 665 670ctc gat ttt cta ctg atg ctt
tgt tcc gtc ggg caa cat tac ccc ggt 2064Leu Asp Phe Leu Leu Met Leu
Cys Ser Val Gly Gln His Tyr Pro Gly 675 680 685ttt gaa acg gat att
cac ggc gcg aaa cag gac gaa gac ggc gtt tac 2112Phe Glu Thr Asp Ile
His Gly Ala Lys Gln Asp Glu Asp Gly Val Tyr 690 695 700cgc gta cga
gtc cta aaa atg gcg gga taa 2142Arg Val Arg Val Leu Lys Met Ala
Gly705 71012713PRTEscherichia coli 12Met Asn Ile Ile Ala Ile Met
Gly Pro His Gly Val Phe Tyr Lys Asp1 5 10 15Glu Pro Ile Lys Glu Leu
Glu Ser Ala Leu Val Ala Gln Gly Phe Gln 20 25 30Ile Ile Trp Pro Gln
Asn Ser Val Asp Leu Leu Lys Phe Ile Glu His 35 40 45Asn Pro Arg Ile
Cys Gly Val Ile Phe Asp Trp Asp Glu Tyr Ser Leu 50 55 60Asp Leu Cys
Ser Asp Ile Asn Gln Leu Asn Glu Tyr Leu Pro Leu Tyr65 70 75 80Ala
Phe Ile Asn Thr His Ser Thr Met Asp Val Ser Val Gln Asp Met 85 90
95Arg Met Ala Leu Trp Phe Phe Glu Tyr Ala Leu Gly Gln Ala Glu Asp
100 105 110Ile Ala Ile Arg Met Arg Gln Tyr Thr Asp Glu Tyr Leu Asp
Asn Ile 115 120 125Thr Pro Pro Phe Thr Lys Ala Leu Phe Thr Tyr Val
Lys Glu Arg Lys 130 135 140Tyr Thr Phe Cys Thr Pro Gly His Met Gly
Gly Thr Ala Tyr Gln Lys145 150 155 160Ser Pro Val Gly Cys Leu Phe
Tyr Asp Phe Phe Gly Gly Asn Thr Leu 165 170 175Lys Ala Asp Val Ser
Ile Ser Val Thr Glu Leu Gly Ser Leu Leu Asp 180 185 190His Thr Gly
Pro His Leu Glu Ala Glu Glu Tyr Ile Ala Arg Thr Phe 195 200 205Gly
Ala Glu Gln Ser Tyr Ile Val Thr Asn Gly Thr Ser Thr Ser Asn 210 215
220Lys Ile Val Gly Met Tyr Ala Ala Pro Ser Gly Ser Thr Leu Leu
Ile225 230 235 240Asp Arg Asn Cys His Lys Ser Leu Ala His Leu Leu
Met Met Asn Asp 245 250 255Val Val Pro Val Trp Leu Lys Pro Thr Arg
Asn Ala Leu Gly Ile Leu 260 265 270Gly Gly Ile Pro Arg Arg Glu Phe
Thr Arg Asp Ser Ile Glu Glu Lys 275 280 285Val Ala Ala Thr Thr Gln
Ala Gln Trp Pro Val His Ala Val Ile Thr 290 295 300Asn Ser Thr Tyr
Asp Gly Leu Leu Tyr Asn Thr Asp Trp Ile Lys Gln305 310 315 320Thr
Leu Asp Val Pro Ser Ile His Phe Asp Ser Ala Trp Val Pro Tyr 325 330
335Thr His Phe His Pro Ile Tyr Gln Gly Lys Ser Gly Met Ser Gly Glu
340 345 350Arg Val Ala Gly Lys Val Ile Phe Glu Thr Gln Ser Thr His
Lys Met 355 360 365Leu Ala Ala Leu Ser Gln Ala Ser Leu Ile His Ile
Lys Gly Glu Tyr 370 375 380Asp Glu Glu Ala Phe Asn Glu Ala Phe Met
Met His Thr Thr Thr Ser385 390 395 400Pro Ser Tyr Pro Ile Val Ala
Ser Val Glu Thr Ala Ala Ala Met Leu 405 410 415Arg Gly Asn Pro Gly
Lys Arg Leu Ile Asn Arg Ser Val Glu Arg Ala 420 425 430Leu His Phe
Arg Lys Glu Val Gln Arg Leu Arg Glu Glu Ser Asp Gly 435 440 445Trp
Phe Phe Asp Ile Trp Gln Pro Pro Gln Val Asp Glu Ala Glu Cys 450 455
460Trp Pro Val Ala Pro Gly Glu Gln Trp His Gly Phe Asn Asp Ala
Asp465 470 475 480Ala Asp His Met Phe Leu Asp Pro Val Lys Val Thr
Ile Leu Thr Pro 485 490 495Gly Met Asp Glu Gln Gly Asn Met Ser Glu
Glu Gly Ile Pro Ala Ala 500 505 510Leu Val Ala Lys Phe Leu Asp Glu
Arg Gly Ile Val Val Glu Lys Thr 515 520 525Gly Pro Tyr Asn Leu Leu
Phe Leu Phe Ser Ile Gly Ile Asp Lys Thr 530 535 540Lys Ala Met Gly
Leu Leu Arg Gly Leu Thr Glu Phe Lys Arg Ser Tyr545 550 555 560Asp
Leu Asn Leu Arg Ile Lys Asn Met Leu Pro Asp Leu Tyr Ala Glu 565 570
575Asp Pro Asp Phe Tyr Arg Asn Met Arg Ile Gln Asp Leu Ala Gln Gly
580 585 590Ile His Lys Leu Ile Arg Lys His Asp Leu Pro Gly Leu Met
Leu Arg 595 600 605Ala Phe Asp Thr Leu Pro Glu Met Ile Met Thr Pro
His Gln Ala Trp 610 615 620Gln Arg Gln Ile Lys Gly Glu Val Glu Thr
Ile Ala Leu Glu Gln Leu625 630 635 640Val Gly Arg Val Ser Ala Asn
Met Ile Leu Pro Tyr Pro Pro Gly Val 645 650 655Pro Leu Leu Met Pro
Gly Glu Met Leu Thr Lys Glu Ser Arg Thr Val 660 665 670Leu Asp Phe
Leu Leu Met Leu Cys Ser Val Gly Gln His Tyr Pro Gly 675 680 685Phe
Glu Thr Asp Ile His Gly Ala Lys Gln Asp Glu Asp Gly Val Tyr 690 695
700Arg Val Arg Val Leu Lys Met Ala Gly705 7101354DNAArtificial
Sequence5'-primer for cadA 13tttgctttct tctttcaata ccttaacggt
atagcgtgaa gcctgctttt ttat 541454DNAArtificial Sequence3'-primer
for cadA 14agatatgact atgaacgtta ttgcaatatt gaatcacgct caagttagta
taaa 541554DNAArtificial Sequence5'-primer for ldc 15ggaggaacac
atgaacatca ttgccattat gggacctgaa gcctgctttt ttat
541653DNAArtificial Sequence3'-primer for ldc 16cgccattttt
aggactcgta cgcggtaaac gccgtccgtc aagttagtat aaa
53172148DNAEscherichia coliCDS(1)..(2145) 17atg aac gtt att gca ata
ttg aat cac atg ggg gtt tat ttt aaa gaa 48Met Asn Val Ile Ala Ile
Leu Asn His Met Gly Val Tyr Phe Lys Glu1 5 10 15gaa ccc atc cgt gaa
ctt cat cgc gcg ctt gaa cgt ctg aac ttc cag 96Glu Pro Ile Arg Glu
Leu His Arg Ala Leu Glu Arg Leu Asn Phe Gln 20 25 30att gtt tac ccg
aac gac cgt gac gac tta tta aaa ctg atc gaa aac 144Ile Val Tyr Pro
Asn Asp Arg Asp Asp Leu Leu Lys Leu Ile Glu Asn 35 40 45aat gcg cgt
ctg tgc ggc gtt att ttt gac tgg gat aaa tat aat ctc 192Asn Ala Arg
Leu Cys Gly Val Ile Phe Asp Trp Asp Lys Tyr Asn Leu 50 55 60gag ctg
tgc gaa gaa att agc aaa atg aac gag aac ctg ccg ttg tac 240Glu Leu
Cys Glu Glu Ile Ser Lys Met Asn Glu Asn Leu Pro Leu Tyr65 70 75
80gcg ttc gct aat acg tat tcc act ctc gat gta agc ctg aat gac ctg
288Ala Phe Ala Asn Thr Tyr Ser Thr Leu Asp Val Ser Leu Asn Asp Leu
85 90 95cgt tta cag att agc ttc ttt gaa tat gcg ctg ggt gct gct gaa
gat 336Arg Leu Gln Ile Ser Phe Phe Glu Tyr Ala Leu Gly Ala Ala Glu
Asp 100 105 110att gct aat aag atc aag cag acc act gac gaa tat atc
aac act att 384Ile Ala Asn Lys Ile Lys Gln Thr Thr Asp Glu Tyr Ile
Asn Thr Ile 115 120 125ctg cct ccg ctg act aaa gca ctg ttt aaa tat
gtt cgt gaa ggt aaa 432Leu Pro Pro Leu Thr Lys Ala Leu Phe Lys Tyr
Val Arg Glu Gly Lys 130 135 140tat act ttc tgt act cct ggt cac atg
ggc ggt act gca ttc cag aaa 480Tyr Thr Phe Cys Thr Pro Gly His Met
Gly Gly Thr Ala Phe Gln Lys145 150 155 160agc ccg gta ggt agc ctg
ttc tat gat ttc ttt ggt ccg aat acc atg 528Ser Pro Val Gly Ser Leu
Phe Tyr Asp Phe Phe Gly Pro Asn Thr Met 165 170 175aaa tct gat att
tcc att tca gta tct gaa ctg ggt tct ctg ctg gat 576Lys Ser Asp Ile
Ser Ile Ser Val Ser Glu Leu Gly Ser Leu Leu Asp 180 185 190cac agt
ggt cca cac aaa gaa gca gaa cag tat atc gct cgc gtc ttt 624His Ser
Gly Pro His Lys Glu Ala Glu Gln Tyr Ile Ala Arg Val Phe 195 200
205aac gca gac cgc agc tac atg gtg acc aac ggt act tcc act gcg aac
672Asn Ala Asp Arg Ser Tyr Met Val Thr Asn Gly Thr Ser Thr Ala Asn
210 215 220aaa att gtt ggt atg tac tct gct cca gca ggc agc acc att
ctg att 720Lys Ile Val Gly Met Tyr Ser Ala Pro Ala Gly Ser Thr Ile
Leu Ile225 230 235 240gac cgt aac tgc cac aaa tcg ctg acc cac ctg
atg atg atg agc gat 768Asp Arg Asn Cys His Lys Ser Leu Thr His Leu
Met Met Met Ser Asp 245 250 255gtt acg cca atc tat ttc cgc ccg acc
cgt aac gct tac ggt att ctt 816Val Thr Pro Ile Tyr Phe Arg Pro Thr
Arg Asn Ala Tyr Gly Ile Leu 260 265 270ggt ggt atc cca cag agt gaa
ttc cag cac gct acc att gct aag cgc 864Gly Gly Ile Pro Gln Ser Glu
Phe Gln His Ala Thr Ile Ala Lys Arg 275 280 285gtg aaa gaa aca cca
aac gca acc tgg ccg gta cat gct gta att acc 912Val Lys Glu Thr Pro
Asn Ala Thr Trp Pro Val His Ala Val Ile Thr 290 295 300aac tct acc
tat gat ggt ctg ctg tac aac acc gac ttc atc aag aaa 960Asn Ser Thr
Tyr Asp Gly Leu Leu Tyr Asn Thr Asp Phe Ile Lys Lys305 310 315
320aca ctg gat gtg aaa tcc atc cac ttt gac tcc gcg tgg gtg cct tac
1008Thr Leu Asp Val Lys Ser Ile His Phe Asp Ser Ala Trp Val Pro Tyr
325 330 335acc aac ttc tca ccg att tac gaa ggt aaa tgc ggt atg agc
ggt ggc 1056Thr Asn Phe Ser Pro Ile Tyr Glu Gly Lys Cys Gly Met Ser
Gly Gly 340 345 350cgt gta gaa ggg aaa gtg att tac gaa acc cag tcc
act cac aaa ctg 1104Arg Val Glu Gly Lys Val Ile Tyr Glu Thr Gln Ser
Thr His Lys Leu 355 360 365ctg gcg gcg ttc tct cag gct tcc atg atc
cac gtt aaa ggt gac gta 1152Leu Ala Ala Phe Ser Gln Ala Ser Met Ile
His Val Lys Gly Asp Val 370 375 380aac gaa gaa acc ttt aac gaa gcc
tac atg atg cac acc acc act tct 1200Asn Glu Glu Thr Phe Asn Glu Ala
Tyr Met Met His Thr Thr Thr Ser385 390 395 400ccg cac tac ggt atc
gtg gcg tcc act gaa acc gct gcg gcg atg atg 1248Pro His Tyr Gly Ile
Val Ala Ser Thr Glu Thr Ala Ala Ala Met Met 405 410 415aaa ggc aat
gca ggt aag cgt ctg atc aac ggt tct att gaa cgt gcg 1296Lys Gly Asn
Ala Gly Lys Arg Leu Ile Asn Gly Ser Ile Glu Arg Ala 420 425 430atc
aaa ttc cgt aaa gag atc aaa cgt ctg aga acg gaa tct gat ggc 1344Ile
Lys Phe Arg Lys Glu Ile Lys Arg Leu Arg Thr Glu Ser Asp Gly 435 440
445tgg ttc ttt gat gta tgg cag ccg gat cat atc gat acg act gaa tgc
1392Trp Phe Phe Asp Val Trp Gln Pro Asp His Ile Asp Thr Thr Glu Cys
450 455 460tgg ccg ctg cgt tct gac agc acc tgg cac ggc ttc aaa aac
atc gat 1440Trp Pro Leu Arg Ser Asp Ser Thr Trp His Gly Phe Lys Asn
Ile Asp465 470 475 480aac gag cac atg tat ctt gac ccg atc aaa gtc
acc ctg ctg act ccg 1488Asn Glu His Met Tyr Leu Asp Pro Ile Lys Val
Thr Leu Leu Thr Pro 485 490 495ggg atg gaa aaa gac ggc acc atg agc
gac ttt ggt att ccg gcc agc
1536Gly Met Glu Lys Asp Gly Thr Met Ser Asp Phe Gly Ile Pro Ala Ser
500 505 510atc gtg gcg aaa tac ctc gac gaa cat ggc atc gtt gtt gag
aaa acc 1584Ile Val Ala Lys Tyr Leu Asp Glu His Gly Ile Val Val Glu
Lys Thr 515 520 525ggt ccg tat aac ctg ctg ttc ctg ttc agc atc ggt
atc gat aag acc 1632Gly Pro Tyr Asn Leu Leu Phe Leu Phe Ser Ile Gly
Ile Asp Lys Thr 530 535 540aaa gca ctg agc ctg ctg cgt gct ctg act
gac ttt aaa cgt gcg ttc 1680Lys Ala Leu Ser Leu Leu Arg Ala Leu Thr
Asp Phe Lys Arg Ala Phe545 550 555 560gac ctg aac ctg cgt gtg aaa
aac atg ctg ccg tct ctg tat cgt gaa 1728Asp Leu Asn Leu Arg Val Lys
Asn Met Leu Pro Ser Leu Tyr Arg Glu 565 570 575gat cct gaa ttc tat
gaa aac atg cgt att cag gaa ctg gct cag aat 1776Asp Pro Glu Phe Tyr
Glu Asn Met Arg Ile Gln Glu Leu Ala Gln Asn 580 585 590atc cac aaa
ctg att gtt cac cac aat ctg ccg gat ctg atg tat cgc 1824Ile His Lys
Leu Ile Val His His Asn Leu Pro Asp Leu Met Tyr Arg 595 600 605gca
ttt gaa gtg ctg ccg acg atg gta atg act ccg tat gct gca ttc 1872Ala
Phe Glu Val Leu Pro Thr Met Val Met Thr Pro Tyr Ala Ala Phe 610 615
620cag aaa gag ctg cac ggt atg acc gaa gaa gtt tac ctc gac gaa atg
1920Gln Lys Glu Leu His Gly Met Thr Glu Glu Val Tyr Leu Asp Glu
Met625 630 635 640gta ggt cgt att aac gcc aat atg atc ctt ccg tac
ccg ccg gga gtt 1968Val Gly Arg Ile Asn Ala Asn Met Ile Leu Pro Tyr
Pro Pro Gly Val 645 650 655cct ctg gta atg ccg ggt gaa atg atc acc
gaa gaa agc cgt ccg gtt 2016Pro Leu Val Met Pro Gly Glu Met Ile Thr
Glu Glu Ser Arg Pro Val 660 665 670ctg gag ttc ctg cag atg ctg tgt
gaa atc ggc gct cac tat ccg ggc 2064Leu Glu Phe Leu Gln Met Leu Cys
Glu Ile Gly Ala His Tyr Pro Gly 675 680 685ttt gaa acc gat att cac
ggt gca tac cgt cag gct gat ggc cgc tat 2112Phe Glu Thr Asp Ile His
Gly Ala Tyr Arg Gln Ala Asp Gly Arg Tyr 690 695 700acc gtt aag gta
ttg aaa gaa gaa agc aaa aaa taa 2148Thr Val Lys Val Leu Lys Glu Glu
Ser Lys Lys705 710 71518715PRTEscherichia coli 18Met Asn Val Ile
Ala Ile Leu Asn His Met Gly Val Tyr Phe Lys Glu1 5 10 15Glu Pro Ile
Arg Glu Leu His Arg Ala Leu Glu Arg Leu Asn Phe Gln 20 25 30Ile Val
Tyr Pro Asn Asp Arg Asp Asp Leu Leu Lys Leu Ile Glu Asn 35 40 45Asn
Ala Arg Leu Cys Gly Val Ile Phe Asp Trp Asp Lys Tyr Asn Leu 50 55
60Glu Leu Cys Glu Glu Ile Ser Lys Met Asn Glu Asn Leu Pro Leu Tyr65
70 75 80Ala Phe Ala Asn Thr Tyr Ser Thr Leu Asp Val Ser Leu Asn Asp
Leu 85 90 95Arg Leu Gln Ile Ser Phe Phe Glu Tyr Ala Leu Gly Ala Ala
Glu Asp 100 105 110Ile Ala Asn Lys Ile Lys Gln Thr Thr Asp Glu Tyr
Ile Asn Thr Ile 115 120 125Leu Pro Pro Leu Thr Lys Ala Leu Phe Lys
Tyr Val Arg Glu Gly Lys 130 135 140Tyr Thr Phe Cys Thr Pro Gly His
Met Gly Gly Thr Ala Phe Gln Lys145 150 155 160Ser Pro Val Gly Ser
Leu Phe Tyr Asp Phe Phe Gly Pro Asn Thr Met 165 170 175Lys Ser Asp
Ile Ser Ile Ser Val Ser Glu Leu Gly Ser Leu Leu Asp 180 185 190His
Ser Gly Pro His Lys Glu Ala Glu Gln Tyr Ile Ala Arg Val Phe 195 200
205Asn Ala Asp Arg Ser Tyr Met Val Thr Asn Gly Thr Ser Thr Ala Asn
210 215 220Lys Ile Val Gly Met Tyr Ser Ala Pro Ala Gly Ser Thr Ile
Leu Ile225 230 235 240Asp Arg Asn Cys His Lys Ser Leu Thr His Leu
Met Met Met Ser Asp 245 250 255Val Thr Pro Ile Tyr Phe Arg Pro Thr
Arg Asn Ala Tyr Gly Ile Leu 260 265 270Gly Gly Ile Pro Gln Ser Glu
Phe Gln His Ala Thr Ile Ala Lys Arg 275 280 285Val Lys Glu Thr Pro
Asn Ala Thr Trp Pro Val His Ala Val Ile Thr 290 295 300Asn Ser Thr
Tyr Asp Gly Leu Leu Tyr Asn Thr Asp Phe Ile Lys Lys305 310 315
320Thr Leu Asp Val Lys Ser Ile His Phe Asp Ser Ala Trp Val Pro Tyr
325 330 335Thr Asn Phe Ser Pro Ile Tyr Glu Gly Lys Cys Gly Met Ser
Gly Gly 340 345 350Arg Val Glu Gly Lys Val Ile Tyr Glu Thr Gln Ser
Thr His Lys Leu 355 360 365Leu Ala Ala Phe Ser Gln Ala Ser Met Ile
His Val Lys Gly Asp Val 370 375 380Asn Glu Glu Thr Phe Asn Glu Ala
Tyr Met Met His Thr Thr Thr Ser385 390 395 400Pro His Tyr Gly Ile
Val Ala Ser Thr Glu Thr Ala Ala Ala Met Met 405 410 415Lys Gly Asn
Ala Gly Lys Arg Leu Ile Asn Gly Ser Ile Glu Arg Ala 420 425 430Ile
Lys Phe Arg Lys Glu Ile Lys Arg Leu Arg Thr Glu Ser Asp Gly 435 440
445Trp Phe Phe Asp Val Trp Gln Pro Asp His Ile Asp Thr Thr Glu Cys
450 455 460Trp Pro Leu Arg Ser Asp Ser Thr Trp His Gly Phe Lys Asn
Ile Asp465 470 475 480Asn Glu His Met Tyr Leu Asp Pro Ile Lys Val
Thr Leu Leu Thr Pro 485 490 495Gly Met Glu Lys Asp Gly Thr Met Ser
Asp Phe Gly Ile Pro Ala Ser 500 505 510Ile Val Ala Lys Tyr Leu Asp
Glu His Gly Ile Val Val Glu Lys Thr 515 520 525Gly Pro Tyr Asn Leu
Leu Phe Leu Phe Ser Ile Gly Ile Asp Lys Thr 530 535 540Lys Ala Leu
Ser Leu Leu Arg Ala Leu Thr Asp Phe Lys Arg Ala Phe545 550 555
560Asp Leu Asn Leu Arg Val Lys Asn Met Leu Pro Ser Leu Tyr Arg Glu
565 570 575Asp Pro Glu Phe Tyr Glu Asn Met Arg Ile Gln Glu Leu Ala
Gln Asn 580 585 590Ile His Lys Leu Ile Val His His Asn Leu Pro Asp
Leu Met Tyr Arg 595 600 605Ala Phe Glu Val Leu Pro Thr Met Val Met
Thr Pro Tyr Ala Ala Phe 610 615 620Gln Lys Glu Leu His Gly Met Thr
Glu Glu Val Tyr Leu Asp Glu Met625 630 635 640Val Gly Arg Ile Asn
Ala Asn Met Ile Leu Pro Tyr Pro Pro Gly Val 645 650 655Pro Leu Val
Met Pro Gly Glu Met Ile Thr Glu Glu Ser Arg Pro Val 660 665 670Leu
Glu Phe Leu Gln Met Leu Cys Glu Ile Gly Ala His Tyr Pro Gly 675 680
685Phe Glu Thr Asp Ile His Gly Ala Tyr Arg Gln Ala Asp Gly Arg Tyr
690 695 700Thr Val Lys Val Leu Lys Glu Glu Ser Lys Lys705 710
715
* * * * *
References